Spheroids and organoids derived from colorectal cancer as tools for in vitro drug screening.

Colorectal cancer (CRC) is a heterogeneous disease. Conventional two-dimensional (2D) culture employing cell lines was developed to study the molecular properties of CRC in vitro. Although these cell lines which are isolated from the tumor niche in which cancer develop, the translation to human model such as studying drug response is often hindered by the inability of cell lines to recapture original tumor features and the lack of heterogeneous clinical tumors represented by this 2D model, differed from in vivo condition. These limitations which may be overcome by utilizing three-dimensional (3D) culture consisting of spheroids and organoids. Over the past decade, great advancements have been made in optimizing culture method to establish spheroids and organoids of solid tumors including of CRC for multiple purposes including drug screening and establishing personalized medicine. These structures have been proven to be versatile and robust models to study CRC progression and deciphering its heterogeneity. This review will describe on advances in 3D culture technology and the application as well as the challenges of CRC-derived spheroids and organoids as a mode to screen for anticancer drugs.

Glycoproteomics-based liquid biopsy: translational outlook for colorectal cancer clinical management in Southeast Asia.

Colorectal cancer (CRC) signifies a significant healthcare challenge in Southeast Asia. Despite advancements in screening approaches and treatment modalities, significant medical gaps remain, ranging from prevention and early diagnosis to determining targeted therapy and establishing personalized approaches to managing CRC. There is a need to expand more validated biomarkers in clinical practice. An advanced technique incorporating high-throughput mass spectrometry as a liquid biopsy to unravel a repertoire of glycoproteins and glycans would potentially drive the development of clinical tools for CRC screening, diagnosis and monitoring, and it can be further adapted to the existing standard-of-care procedure. Therefore this review offers a perspective on glycoproteomics-driven liquid biopsy and its potential integration into the clinical care of CRC in the southeast Asia region.

Identification of AOC3 and LRRC17 as Colonic Fibroblast Activation Markers and Their Potential Roles in Colorectal Cancer Progression.

BACKGROUND: Accumulation of cancer-associated fibroblasts (CAFs) in the tumor stroma is linked to poor prognosis in colorectal cancer (CRC). CAF-cancer cell interplay, facilitated by secretomes including transforming growth factor-beta 1 (TGF-ß1), supports fibroblast activation, drives colorectal carcinogenesis, and contributes to CRC aggressive phenotypes. Although widely used, traditional CAF biomarkers are found to have heterogeneous and non-specific expression. Amine oxidase copper containing 3 (AOC3) and leucine-rich repeat-containing 17 (LRRC17) have been reported to be emerging markers of myofibroblasts. AIM: Our objective was to investigate the potential of AOC3 and LRRC17 as biomarkers for fibroblast activation thus predicting their roles in CRC progression. METHODS: Immunofluorescence (IF) staining of AOC3 and LRRC17 was performed on myofibroblast line (CCD-112CoN), primary fibroblasts from colorectal tumor (CAFs), and adjacent normal tissue (normal fibroblasts-NFs). SW620 (epithelial CRC cell line) was used as a control.  Conventional CAF biomarker (alpha-smooth muscle actin - α-SMA) was included in the IF analysis. Fluorescence intensity was compared between groups using ImageJ software. Proliferation and contractility of treated cells were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and collagen gel contraction assays, respectively. Fibroblast contraction under TGF-ß1 treatment was compared to those treated with complete medium (addition of 10% serum) and serum free (SF) medium. RESULTS: Positive AOC3, LRRC17, and α-SMA expression were observed in colonic fibroblasts, more prominent in CAFs, whereas negative staining was found in SW620. Significant downregulation of AOC3, and upregulations in LRRC17 and α-SMA expression was found in TGF-ß1-treated fibroblasts compared to SF medium treatment (p-value<0.05). All fibroblasts exhibited higher proliferation in complete medium and under treatment with conditioned medium from SW620 than SF medium. Significant contraction of NFs was recorded in complete medium and TGF-ß1 (p-value<0.01). CONCLUSION: Our results demonstrate AOC3 and LRRC17 as the potential markers of CAF activation which promote CRC progression.

KRAS Mutational Profiles among Colorectal Cancer Patients in the East Coast of Peninsular Malaysia.

BACKGROUND: KRAS is a key driver gene in colorectal carcinogenesis. Despite this, there are still limited data on the mutational status of KRAS amongst colorectal cancer (CRC) patients in Malaysia. In the present study, we aimed to analyze the KRAS mutational profiles on codons 12 and 13 amongst CRC patients in Hospital Universiti Sains Malaysia, Kelantan, located on the East Coast of Peninsular Malaysia. METHODS: DNA were extracted from formalin-fixed, paraffin-embedded tissues obtained from 33 CRC patients diagnosed between 2018 and 2019. Amplifications of codons 12 and 13 of KRAS were conducted using conventional polymerase chain reaction (PCR) followed by Sanger sequencing. RESULTS: Mutations were identified in 36.4% (12/33) of patients, with G12D (50%) being the most frequent single-point mutation observed, followed by G12V (25%), G13D (16.7%), and G12S (8.3%). No correlation was found between mutant KRAS and location of the tumor, staging, and initial carcinoembryonic antigen (CEA) level. CONCLUSION: Current analyses revealed that a significant proportion of CRC patients in the East Coast of Peninsular Malaysia have KRAS mutations, where this frequency is higher compared to those in the West Coast. The findings of this study would serve as a precursor for further research that explores KRAS mutational status and the profiling of other candidate genes among Malaysian CRC patients.

Dynamic Co-Evolution of Cancer Cells and Cancer-Associated Fibroblasts: Role in Right- and Left-Sided Colon Cancer Progression and Its Clinical Relevance.

Cancer is a result of a dynamic evolutionary process. It is composed of cancer cells and the tumour microenvironment (TME). One of the major cellular constituents of TME, cancer-associated fibroblasts (CAFs) are known to interact with cancer cells and promote colorectal carcinogenesis. The accumulation of these activated fibroblasts is linked to poor diagnosis in colorectal cancer (CRC) patients and recurrence of the disease. However, the interplay between cancer cells and CAFs is yet to be described, especially in relation to the sidedness of colorectal carcinogenesis. CRC, which is the third most commonly diagnosed cancer globally, can be classified according to the anatomical region from which they originate: left-sided (LCRC) and right-sided CRC (RCR). Both cancers differ in many aspects, including in histology, evolution, and molecular signatures. Despite occurring at lower frequency, RCRC is often associated with worse diagnosis compared to LCRC. The differences in molecular profiles between RCRC and LCRC also influence the mode of treatment that can be used to specifically target these cancer entities. A better understanding of the cancer cell-CAF interplay and its association with RCRC and LRCR progression will provide better insight into potential translational aspects of targeted treatment for CRC.

Gene Therapy Targeting p53 and KRAS for Colorectal Cancer Treatment: A Myth or the Way Forward?

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy worldwide and is responsible as one of the main causes of mortality in both men and women. Despite massive efforts to raise public awareness on early screening and significant advancements in the treatment for CRC, the majority of cases are still being diagnosed at the advanced stage. This contributes to low survivability due to this cancer. CRC patients present various genetic changes and epigenetic modifications. The most common genetic alterations associated with CRC are p53 and KRAS mutations. Gene therapy targeting defect genes such as TP53 (tumor suppressor gene encodes for p53) and KRAS (oncogene) in CRC potentially serves as an alternative treatment avenue for the disease in addition to the standard therapy. For the last decade, significant developments have been seen in gene therapy for translational purposes in treating various cancers. This includes the development of vectors as delivery vehicles. Despite the optimism revolving around targeted gene therapy for cancer treatment, it also has various limitations, such as a lack of availability of related technology, high cost of the involved procedures, and ethical issues. This article will provide a review on the potentials and challenges of gene therapy targeting p53 and KRAS for the treatment of CRC.

In Vitro Analyses of Interactions Between Colonic Myofibroblasts and Colorectal Cancer Cells for Anticancer Study.

BACKGROUND/AIM: Interactions between colorectal cancer (CRC) cells and myofibroblasts govern many processes such as cell growth, migration, invasion and differentiation, and contribute to CRC progression. Robust experimental tests are needed to investigate the nature of these interactions for future anticancer studies. The purpose of the study was to design and validate in vitro assays for studying the communication between myofibroblasts and CRC epithelial cell lines. MATERIALS AND METHODS: The influence of co-culture of myofibroblasts and CRC cell lines is discussed using various in vitro assays including direct co-culture, transwell assays, Matrigel-based differentiation and cell invasion experiments. RESULTS: The results from these in vitro assays clearly demonstrated various aspects of the crosstalk between myofibroblasts and CRC cell lines, which include cell growth, differentiation, migration and invasion. CONCLUSION: The reported in vitro assays provide a basis for investigating the factors that control the myofibroblast-epithelial cell interactions in CRC in vivo.

Cancer-associated fibroblasts of colorectal cancer and their markers: updates, challenges and translational outlook.

Accumulation of cancer-associated fibroblasts (CAFs) in the tumor microenvironment is associated with poor prognosis and recurrence of colorectal cancer (CRC). Despite their prominent roles in colorectal carcinogenesis, there is a lack of robust and specific markers to classify the heterogeneous and highly complex CAF populations. This has resulted in confusing and misleading definitions of CAFs in cancer niche. Advancements in molecular biology approaches have open doors to reliable CAF marker detection methods in various solid tumors. These discoveries would contribute to more efficient screening, monitoring and targeted therapy of CRC thus potentially will reduce cancer morbidity and mortality rates. This review highlights current scenarios, dilemma, translational potentials of CAF biomarker and future therapeutic applications involving CAF marker identification in CRC.

Single-cell analysis on stromal fibroblasts in the microenvironment of solid tumours.

Besides malignant cells, the tumour microenvironment consists of various stromal cells such as cancer-associated fibroblasts (CAFs) and myofibroblasts. Accumulation of heterogeneous populations of stromal cells in solid tumours is associated with lower survival rates and cancer recurrence in patients. Certain limitations presented by conventional experimental designs and techniques in cancer research have led to poor understanding of the fundamental basis of cancer niche. Recent developments in single-cell techniques allow more in-depth studies of the tumour microenvironment. Analyses at the single-cell level enables the detection of rare cell types, characterization of intra-tumour cellular heterogeneity and analysis of the lineage output of malignant cells. This subsequently, provides valuable insights on better diagnostic methods and treatment avenues for cancer. This review explores the recent advancements and applications of single-cell technologies in cancer research pertaining to the study of stromal fibroblasts in the microenvironment of solid tumours.

Effects of Perivitelline Fluid Obtained from Horseshoe Crab on The Proliferation and Genotoxicity of Dental Pulp Stem Cells.

OBJECTIVE: Perivitelline fluid (PVF) of the horseshoe crab embryo has been reported to possess an important role during embryogenesis by promoting cell proliferation. This study aims to evaluate the effect of PVF on the proliferation, chromosome aberration (CA) and mutagenicity of the dental pulp stem cells (DPSCs). MATERIALS AND METHODS: This is an in vitro experimental study. PVF samples were collected from horseshoe crabs from beaches in Malaysia and the crude extract was prepared. DPSCs were treated with different concentrations of PVF crude extract in an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (cytotoxicity test). We choose two inhibitory concentrations (IC50 and IC25) and two PVF concentrations which produced more cell viability compared to a negative control (100%) for further tests. Quantitative analysis of the proliferation activity of PVF was studied using the AlamarBlue®assay for 10 days. Population doubling times (PDTs) of the treatment groups were calculated from this assay. Genotoxicity was evaluated based on the CA and Ames tests. Statistical analysis was carried out using independent t test to calculate significant differences in the PDT and mitotic indices in the CA test between the treatment and negative control groups. Significant differences in the data were P<0.05. RESULTS: A total of four PVF concentrations retrieved from the MTT assay were 26.887 mg/ml (IC50), 14.093 mg/ml (IC25), 0.278 mg/ml (102% cell viability) and 0.019 mg/ml (102.5% cell viability). According to the AlamarBlue®assay, these PVF groups produced comparable proliferation activities compared to the negative (untreated) control. PDTs between PVF groups and the negative control were insignificantly different (P>0.05). No significant aberrations in chromosomes were observed in the PVF groups and the Ames test on the PVF showed the absence of significant positive results. CONCLUSION: PVF from horseshoe crabs produced insignificant proliferative activity on treated DPSCs. The PVF was non-genotoxic based on the CA and Ames tests.

Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.

BACKGROUND: Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth and proliferation. The aim of this study was to evaluate the effect of royal jelly as an alternative to fetal bovine serum (FBS) in cell culture using cell proliferation assays and live cell imaging. MATERIALS AND METHODS: MRC-5 cells were treated with various concentrations of royal jelly extract in MTT assay. The control groups were comprised of Alpha-Minimal Essential Medium (α-MEM) alone and α-MEM with 10% FBS. Subsequently, the cell proliferation was studied for 10 days using Alamar Blue assay and live cell imaging from 48 to 72 h. The population doubling time (PDT) was determined using trypan blue assay after live cell imaging. RESULTS: In MTT assay, 0.156 and 0.078 mg/ml of royal jelly produced higher cell viability compared to positive control group but were not significantly different (P > 0.05). In the Alamar Blue assay, 0.156 and 0.078 mg/ml of royal jelly produced greater percentage of reduction at day 3 even though no significant difference was found (P > 0.05). Based on live cell imaging, the PDT for positive, negative, 0.156 and 0.078 mg/ml of royal jelly groups were 29.09, 62.50, 41.67 and 41.67 h respectively. No significant difference was found in the PDT between all the groups (P > 0.05). CONCLUSION: Royal jelly does not exhibit similar ability like FBS to facilitate cell growth under the present test conditions.

Genotoxicity evaluation of dental restoration nanocomposite using comet assay and chromosome aberration test.

Nanocomposite is used as a dental filling to restore the affected tooth, especially in dental caries. The dental nanocomposite (KelFil) for tooth restoration used in this study was produced by the School of Dental Sciences, Universiti Sains Malaysia, Malaysia and is incorporated with monodispersed, spherical nanosilica fillers. The aim of the study was to determine the genotoxic effect of KelFil using in vitro genotoxicity tests. The cytotoxicity and genotoxicity of KelFil was evaluated using MTT assay, comet assay and chromosome aberration tests with or without the addition of a metabolic activation system (S9 mix), using the human lung fibroblast cell line (MRC-5). Concurrent negative and positive controls were included. In the comet assay, no comet formation was found in the KelFil groups. There was a significant difference in tail moment between KelFil groups and positive control (p < 0.05). Similarly, no significant aberrations in chromosomes were noticed in KelFil groups. The mitotic indices of treatment groups and negative control were significantly different from positive controls. Hence, it can be concluded that the locally produced dental restoration nanocomposite (KelFil) is non-genotoxic under the present test conditions.