title: The molecular mechanism of tumour budding and its relationship with tumour microenvironment in colorectal cancer
Creators: name: Hatthakarnkul, Phimmada
Publisher: name: University of Glasgow
Year: 2024

description: Summary
Colorectal cancer (CRC) is the third most diagnosed cancer and the second lethal disease worldwide (1). CRC development has recently been well-documented, and the screening program has been shown to improve patient outcomes and survival due to the early detection of the disease (2, 3). However, some patients still experienced disease metastasis with a 5 year survival of only 12% (4). Recent studies have focused on identifying prognostic biomarkers that can predict the adverse outcomes in CRC patients (5).

One promising factor that has recently been reported is tumour budding (TB). TB, the single or up to four tumour cells found at the invasive tumour area, is now a well-known prognostic independent biomarker in many solid cancers including CRC (6). Patients with high TB phenotype experienced a poor outcome with an incidence of disease recurrence and metastasis (7). In 2016, the international tumour budding consensus conference (ITBCC) was held and agreed to set up the criteria for TB assessment and suggested to include TB status in a routine clinical report (8). Since then, multiple studies have investigated the prognostic role of TB not only in CRC but also in other solid cancers such as pancreatic (9), breast (10, 11), head and neck (12) and lung (13) cancer.

Although TB has a strong prognostic value, few studies investigated its underlying mechanism and how it may relate to adverse features and disease metastasis in CRC. It has been hypothesised that TB could undergo epithelial-mesenchymal transition (EMT), thereby, allowing cells to escape from the main tumour and promote metastasis (14, 15). However, some studies argued that TB may only undergo partial EMT and there is another tumour-related signalling involved in its formation and induction of the metastasis (16, 17). Moreover, some studies have reported an inverse correlation between TB and cytotoxic T cells which could suggested an immunosuppressive role of TB leading to disease metastasis in CRC (18-20).

Until now, there has been little understanding of the underlying mechanism of TB and its relationship with the tumour microenvironment in CRC (21). This thesis aims to unravel the molecular mechanism of TB to identify the potential tumour signalling that drives TB formation and how TB is associated with the immune profile at the invasive edge of the tumour. To investigate this, TB status in CRC patients has been identified according to the TB assessment criteria from ITBCC. After that, bulk transcriptomic RNA (n=787) was used to identify tumour-related signalling expressed in tumours with high TB phenotype compared to low TB group. In addition, regional bulk spatial transcriptomic (GeoMx) (n=12) was performed to identify gene expression within the region of interested (ROI), the classification of tumour and stromal areas using specific protein mask (PanCK+/-) was done. This allows the identification of the potential genes related to both budding tumour cells and the surrounded tumour microenvironment between tumours with low and high TB profile as well as the different area of interest (AOI); tumour core, invasive edge, distant stromal area, within the same tumours. The results from GeoMx were later validated in a TMA of the full CRC cohort (n=787), using immunohistochemistry, to verify the translation from RNA to protein. Of these, cyclinD1 expression within TB was identified as a promising prognostic value in CRC patients. Additionally, multiplex immunofluorescence (mIF) using immune panels (lymphocytes and myeloid cells) were also performed to investigate the immune profile within the invasive budding area. Results showed a high density of regulatory and low cytotoxic T cells within the invasive compared to further stromal area of tumours with high TB. Nearest neighbour analysis also showed that TB tend to have a closer distant to regulatory cells as well as pan-macrophages. This finding suggested that TB may have a possible interaction with the surrounding immune cells leading to an alteration of the microenvironment to help it thrive and invade other parts of the body.

To investigate if TB formation can be observed within an in vitro setting, CRC spheroids were cultured. The induction of TNF-α and TGF-β were shown to stimulate more TB formation in CRC spheroids and that cyclinD1 expression within the TB was higher in treated spheroids compared to control groups. Moreover, mouse AKPT organoids showed an increased in roundness, which indicates disruption in the formation of TB, in treated compared to control groups. These results suggested that cyclinD1 expressed within TB could have a potential role as a prognostic marker and may be used as a biomarker for TB formation.

In summary, data from this thesis have demonstrated potential biomarkers of TB and the relationship with tumour microenvironment in CRC. This will help understand the underlying mechanism of TB, and how they might interact with the surrounding microenvironment and could also pave the way for a future target therapeutic approach in CRC