The term inflammatory bowel disease (IBD) encompasses ulcerative colitis (UC) and Crohn’s disease (CD), and describes an immune mediated inflammation of the gut. UC typically presents as mucosally based proctitis extending proximally in a contiguous fashion and involving the colon, whereas CD may manifest at any location in the gastrointestinal tract, causing transmural inflammation and ‘skip lesions’ with regions of macroscopically ‘normal’ bowel intervening. Chronic colonic inflammation is associated with an increased risk of colorectal cancer (CRC); this is proportional to the extent, degree and duration of inflammation (1–3). There is some evidence to suggest that colitis associated CRC (CAC) are more likely to present with higher histologic grade of CRC and have worse overall survival (4–6). CRC is a significant contributor to the overall morbidity and mortality in IBD, with 14% of deaths attributed to CRC (7, 8).

Surveillance colonoscopy is recommended for patients with significant chronic colonic inflammation based on clinically based, individualised risk stratification (9), however, repeated colonoscopy is expensive, invasive and poor at risk-prediction, resulting in both over- and under-treatment. This is in part due to a lack of ability to distinguish those who will progress from inflammation or low-grade dysplasia (LGD) to invasive CRC (progressors), and those ‘non-progressors’ whose disease will follow a more indolent course (10–12). Identifying and validating potential biomarkers that reliably predict which patients will progress to invasive disease would allow a more tailored surveillance and management plan to be devised for individuals and be critical to clinical decision making.

The identification of biomarkers requires a thorough understanding of the pathogenesis and molecular changes underpinning colitis-associated CRC (CAC). Several pathways and mutations identified in sporadic CRC are seen in CAC, however, there are significant differences that imply a distinct inflammatory-mediated process driving CAC (13).

To date, no biomarkers have been identified that are sufficiently consistent, reliable and clinically relevant to have progressed into clinical practice. Molecular profiling of CAC has suggested alterations in gene expression in pathways related to i) transcription and DNA repair; ii) cell cycle and growth; iii) cellular metabolism; iv) cellular communication and v) signal transduction (14). Loss of gene expression, especially of immune response proteins, is associated with neoplastic progression (14).

Most techniques used to quantify gene expression, so far, have been spatially agnostic, limiting the interpretability of the findings. More recently, digital spatial profiling (DSP) techniques have been developed that allow the quantification and localisation of gene expression, facilitating a better understanding of the significance of these changes within the tissue architecture (15). This is particularly useful in the context of our current paradigm of CAC development, which suggests a complex interaction between the mucosa, its microbiome and the immune microenvironment (16).

The aim of this study was to utilise DSP to characterise the molecular and cellular changes associated with transformation of normal and inflamed tissue to dysplasia and invasive CAC, in order to derive potential biomarkers to distinguish risk of progression at an earlier stage. The secondary objective was to investigate changes in the immune-cell populations associated with CAC, in order to better understand the role of the immune microenvironment in malignant transformation.

Recent studies have shown that the organisation and structure of cancer cells within a tumour, known as tumour microenvironment, plays a significant role in the development and progression of cancer. This includes the spatial distribution of different cell types within the tumour, as well as the formation of specific structures such as blood vessels and immune cells. Understanding the tumour architecture can provide important insights into the biology of a cancer, and may help to improve diagnosis, treatment, and prognosis. Previous research techniques have described quantitative differences in protein and gene expression in colitis-associated CRC (CAC), however, most have lacked the capacity to localise the site of expression, limiting the interpretation of this data. Novel spatial profiling techniques, such as Digital Spatial Profiling (DSP), allow a more nuanced insight into the alterations of gene expression and dysregulation of cellular pathways in CAC, which is critical to the development of clinically relevant biomarkers for prognostication in IBD. This study identified significant differences in gene expression as well as immune-cell infiltration between normal, inflamed, dysplastic and CAC tissue. Additionally, the normal and inflamed tissue of progressors and non-progressors show significantly different gene expression.

Unsurprisingly, there were several genes that were differentially expressed across tissue types. The most marked cluster comprised of collagen genes associated with tissue remodelling (COL1A1, COL3A1 and COL6A3); these show steady increase in expression across normal, inflamed, dysplastic and cancer tissue. This is consistent with existing literature in non-IBD CRC, and demonstrates the integral role of the collagen in the tumour microenvironment, including interfacing with the immune population and promoting metastasis (21, 22).

Overexpression of the proto-oncogene, MYC, was observed in dysplastic and cancer tissue, similar to a study of 47 CAC patients, where a higher rate of MYC amplification was reported compared to sporadic CRC, highlighting its importance to the CAC pathway, specifically (23). MYC is a transcription factor responsible for modulating cellular proliferation, and orchestrating changes in the tumour microenvironment, including angiogenesis and immune response. MYC is a key factor in Wnt pathway activation, which is an early event in CRC development. While in sporadic CRC, the Wnt pathway is most commonly activated through mutations in the APC gene, these mutations are seen less frequently and later in CAC. Overexpression of MYC suggests a mechanism by which the Wnt pathway may be activated in the absence of a primary APC mutation, with a similar oncogenic effect.

The role of mucins and the significance of changes in mucin gene expression has been controversial in both IBD and CRC (24, 25). MUC4 codes for one of the mucins contributing to the protective mucous layer lining the small and large bowel, and its over-expression has been associated with both poorer outcome (26) and enhancement of anti-tumour response (27). MUC4 ^-/- mice showed an attenuated colitic response to dextran sodium sulphate and a reduced tumour burden compared to wild-type mice suggesting a role for MUC4 in inflammation and progression to neoplasia, however, the discrepancy between studies implies it is a complex relationship (28). We found a significantly lower expression of MUC4 in dysplastic and cancer tissue, suggesting that progressive loss of MUC4 may be implicated in the development of CAC.

In order to identify potential early biomarkers of disease progression, a comparison of normal and inflamed tissue from ‘progressors’ and ‘non-progressors’ was performed. The LYZ gene, coding for lysozyme, an enzyme produced to digest bacterial cell walls, was dramatically under expressed in the progressors in contrast to the non-progressors, and this under-expression was driven by the immune-cell components in the tissue microenvironment. Lysozyme is normally produced by the Paneth cells of the healthy caecum and right-sided colon. In IBD, Paneth cell metaplasia is seen in the remaining colon with an associated increase in lysozyme production (29). Both faecal and serum lysozyme have been proposed as markers of disease severity in IBD (30). More recently, a series of murine experiments have demonstrated an exaggerated colitic response associated with the ectopic expression of lysozyme; in addition, the authors showed that loss of LYZ expression affected the immune populations through its effect on the mucosal microbiome (31).

The critical role of lysozyme in the regulation of the microbiome may provide a mechanism through which it may drive chronic inflammation and neoplastic transformation. Lysozyme has been shown to have inhibitory effects on tumour proliferation through both direct and indirect modulation of host immunity, inducing interferon production, as well as interruption of both interleukin and S100A6 signalling. In vitro co-culture with lysozyme has led to inhibition of proliferation in a variety of cancer cell lines (32, 33). This suggests that the loss of expression impairs mucosal immunity and promotes tumour development, and highlights the potential use of LYZ as a biomarker of progression to CAC.

The oncogene, CD24, was consistently over-expressed in progressors compared to non-progressors in this study. It is a small cell-surface glycoprotein, expressed on a number of immune cells, that plays a role in both signal transduction, immune regulation, and cellular adhesion. It has been implicated in early-stage carcinogenesis, as well as poorer prognosis tumours (34). In the APC^Min/+ mouse model of colorectal carcinogenesis, CD24 knock-out reduces tumour development in a dose-dependent way (reduction in burden with heterozygous loss and no tumour production with homozygous knock-out) (35). This is a promising biomarker to discriminate progressors from non-progressors and even has potential as a therapeutic target; antibodies to CD24 have been used in in in vitro and murine models to suppress invasion and proliferation of tumour cells through interruption of STAT3 phosphorylation (36), and Ras/BCL-2 pathway down-regulation (37, 38).

The importance of the tumour immune microenvironment is of particular significance to CAC, given the proposed role of chronic inflammation in the development of neoplasia. Analysis of immune cell populations suggests significant heterogeneity, with clustering of different possible sub-types within the CAC group. It is likely that this represents the differences in immune activation and CAC may in fact encompass a diverse collection of distinct CRC pathways and phenotypes. Similarly, a recent single-cell RNA-sequencing and spatial transcriptomics study by Garrido-Trigo et al. identified macrophage and neutrophil components to have the highest heterogeneity among IBD patients (39). Although this study did not include patients with progressive disease, diversity in immune and stromal cell components points to patient-dependent factors that may explain the heterogeneity seen in IBD and be important for progression to CAC. We also identified several genes associated with immune activity that were differentially expressed between progressors and non-progressors, including CCL25. In normal, non-inflamed tissue, the chemokine CCL25 is expressed in the small bowel and not typically in the colon and plays a role in T- and B-cell recruitment. CCL25 expression has been shown to correlate with the presence and severity of colitis (40). In our study, progressors showed a reduced expression of CCL25 compared to those that did not progress, suggesting a difference in lymphocyte recruitment in the development of CAC, compared to patients with IBD only. Similarly, IL-7 receptor (IL-7R) and CCL18 gene expression was also reduced in progressors compared to non-progressors. Higher levels of expression of IL-7R are associated with non-responsiveness to anti-TNF therapy in IBD and hence, higher inflammatory burden (36), while CCL18 is a chemokine that induces differentiation of T-cells towards a ‘pro-tumour’ type macrophage and facilitates matrix remodelling and immune evasion (41, 42). While the reduced expression of these pro-inflammatory mediators may reflect the higher inflammatory burden in the study population undergoing colectomy for medically refractory colitis, compared to the patient group who underwent colectomy for dysplasia or cancer, it may also represent underlying differences in immune activation between the two patient groups.

The T-cell markers, CD3 and CD6, showed lower gene expression in the normal and inflamed tissue from progressors compared to non-progressors. Previous immune profiling studies have shown a reduction in CD3⁺ and CD8⁺ cells in CAC compared to sporadic CRC (43). In contrast, a study comparing microsatellite stable CAC and sporadic CRC demonstrated an increase in CD3⁺ and CD8⁺ cells in CAC, but without an associated increase in tumour cell apoptosis (44). Our study uses different comparators both in terms of the tissue used (normal/inflamed), and the comparator groups (progressors/non-progressors), making it difficult to directly compare the data. However, it is likely that inherent differences in the immune populations and their function contribute to the risk of progression.

The major limitation of this feasibility study is the retrospective nature and limited numbers of patients available. Use of colectomy specimens has meant that there is an inherent difference in the population groups due to the different indications for surgery. This has been compensated for to some degree with the use of histologically normal tissue from the different patients. While this study has demonstrated the potential of DSP to identify biomarkers from a small cohort and the importance of the spatial context of biomarker discovery, validation studies are necessary to confirm the preliminary findings from this small cohort.

This study has demonstrated key differences in gene expression across tissue types and between progressors and non-progressors. The future potential of this study findings is in the identification and confirmation of early changes that predict progression to dysplasia and cancer, enabling better risk stratification, more accurately informed clinical decision making and future therapeutic target development. Future studies should focus on prospective validation of these findings to confirm and better develop understanding of these findings. This is an area of clinical need, where an accurate biomarker would have the potential to revolutionise clinical management for these patients.

The authors acknowledge that the data presented in this study must be deposited and made publicly available in an acceptable repository, prior to publication. Frontiers cannot accept a article that does not adhere to our open data policies.

The studies involving humans were approved by Health and disability ethics committees of New Zealand. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

TG was involved in study design, patient selection, data acquisition, manuscript writing and editing. SW was responsible for data analyses and bioinformatics MW was responsible for histopathological assessment and sample selection TE was involved in study design and manuscript writing and editing NW was involved in study design, methodology and data analyses RP was involved in study design, data analyses, manuscript writing and editing