Background:Tumour budding, described as the presence of single cells or small clusters of up to five tumour cells at the invasive margin, is established as a prognostic marker in colorectal carcinoma. In the present study, we aimed to investigate the molecular signature of tumour budding cells and the corresponding tumour bulk.Methods:Tumour bulk and budding areas were microdissected and processed for RNA-sequencing. As little RNA was obtained from budding cells, a special low-input mRNA library preparation protocol was used. Gene expression profiles of budding as compared with tumour bulk were investigated for established EMT signatures, consensus molecular subtype (CMS), gene set enrichment and pathway analysis.Results:A total of 296 genes were differentially expressed with an FDR <0.05 and a twofold change between tumour bulk and budding regions. Genes that were upregulated in the budding signature were mainly involved in cell migration and survival while downregulated genes were important for cell proliferation. Supervised clustering according to an established EMT gene signature categorised budding regions as EMT-positive, whereas tumour bulk was considered EMT-negative. Furthermore, a shift from CMS2 (epithelial) to CMS4 (mesenchymal) was observed as tumour cells transit from the tumour bulk to the budding regions.Conclusions:Tumour budding regions are characterised by a phenotype switch compared with the tumour bulk, involving the acquisition of migratory characteristics and a decrease in cell proliferation. In particular, most tumour budding signatures were EMT-positive and switched from an epithelial subtype (CMS2) in the tumour bulk to a mesenchymal subtype (CMS4) in budding cells.

Background:Microsatellite instability (MSI) accounts for 15% of all colorectal tumours. Several specific clinicopathologicals (e.g., preference for the proximal colon over the distal colon, improved prognosis and altered response to chemotherapeutics) are described for this subset of tumours. This study aimed to analyse morphological, inflammatory and angiogenic features of MSI vs microsatellite stable (MSS) tumours.Methods:Twenty-seven MSS and 29 MSI, TNM stage matched, colorectal tumours were selected from the archive of the Department of Pathology, UZ Leuven. Morphology was analysed on haematoxylin–eosin sections. Immunohistochemistry for CD3, CD4, CD8, CD20 and CD68 was used to map tumour infiltration in both a digital and traditional microscope-based manner for all distinct morphological components of the tumour. CD31 immunostains were performed to assess angiogenesis.Results:Morphological tumour heterogeneity was a marked feature of MSI tumours, occurring in 53% of the cases as compared with 11% of the MSS tumours (P<0.001). Digital immune quantification showed an increased number of tumour-infiltrating cytotoxic T-lymphocytes (CD8+) in MSI compared with MSS tumours for both the tumour (P=0.02) and peritumoural area (P=0.03). Traditional microscope-based quantification confirmed these results (P<0.001 for both) and, in addition, revealed large numbers of CD68+ macrophages in the peritumoural area of MSI cancers (P=0.001). Moreover, traditional microscope-based analysis was able to distinguish between lymphocytes directly infiltrating the tumoural glands (intra-epithelial) and those infiltrating only the neoplastic stroma around the glands (intratumoural). Quantification showed high numbers of intra-epithelial CD3+, CD4+, CD8+, CD20+ and CD68+ cells in MSI compared with MSS cancers (P<0.001, P=0.01, P<0.001, P<0.001 and P=0.006, respectively). Higher microvessel density (MVD) was observed in MSI tumours compared with their MSS counterpart.Conclusions:Mixed morphology, reflecting tumour heterogeneity, is an important feature of MSI tumours and may have both diagnostic and therapeutic impact. The inflammatory reaction also presented with significant differences in MSI vs MSS colorectal tumours. MSI cancers showed mainly infiltration by cytotoxic T-cells in both the tumour and the close border around the tumour, as well as increased intra-epithelial infiltration in contrast to MSS tumours. The type of immune cell and the compartment it resides in (intratumoural or intra-epithelial) depend both on MSI status and morphology. Finally, MSI tumours showed a higher angiogenic capacity represented by an increased MVD, hinting for possible therapeutic consequences.

Budding is a process during which individual or small clusters of up to five tumour cells detach from the main tumour mass and invade into the surrounding stroma. In colorectal cancer, this feature is observed in 20-40% of cases and is associated with lymphovascular invasion, lymph node and distant metastases, and poor prognosis. A variety of scoring systems for budding have been proposed but so far a gold standard is lacking, hampering implementation of a budding score in guidelines for pathological examination of colorectal cancer. Furthermore, little is known about the mechanisms which cause tumour cells to detach from the main tumour mass and obtain increased invasive potential. In this review, we present an overview of tumour budding including its definition, scoring systems, prognostic relevance and biological mechanisms involved.

In our case series, loss of FOXP1 was associated with reduced survival rates in CRC tissue. Also, FOXP1 affects proliferation and inflammatory reaction in colorectal neoplasia.

The Leidenfrost nanochemistry synthesis of gold colloids was introduced in this Journal as an advanced method to synthesize gold nanoparticles during an undergraduate nanochemistry research project. When trying to repeat these experiments in the laboratory, we experienced difficulties in the synthesis of the gold nanoparticles since the droplets undergoing the Leidenfrost phenomenon were evaporated without changing color to the distinctive red-purple of gold nanoparticles. Therefore, we would like to suggest an alternative procedure to perform this gold nanoparticle synthesis through the Leidenfrost nanochemistry phenomenon by using honey instead of trisodium citrate and adjusting the pH with sodium hydroxide.