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HCT116 Cell Line: A Pillar in Colorectal Cancer Research

The HCT116 cell line serves as a cornerstone in the exploration of colorectal cancer, offering invaluable insights into the disease's pathogenesis and potential therapeutic avenues. Renowned for its utility in cancer research and pharmacological assessments, HCT116 facilitates pivotal studies on tumor behavior and drug efficacy.

Origins and Fundamental Characteristics of HCT116 Cells

Understanding the roots and basic traits of HCT116 cells, such as their morphological features, genetic makeup, and cellular dimensions, is essential for researchers embarking on studies using this cell line.

Provenance and Genetic Landscape: Originating from the colon of a 48-year-old Caucasian male diagnosed with colorectal cancer, HCT116 cells are notable for a mutation in codon 13 (G13D) of the KRAS gene, part of the RAS/RAF/MEK/ERK signaling pathway. This particular mutation is pivotal in the oncogenic transformation of these cells, underlining their relevance in cancer research.

Morphology and Growth Characteristics: Exhibiting an epithelial-like morphology, HCT116 cells typically grow in monolayer cultures but can also form spheroids with diameters of 150-400 µm. This adaptability in growth patterns underscores their versatility in various experimental setups.

Chromosomal Profile: The chromosomal composition of HCT116 cells is near-diploid, with approximately 70% of the cell population harboring 45 chromosomes. Notably, there's a recurrent amplification in the long arms of chromosomes 8, 10, 16, and 17, whereas chromosome Y is absent, contributing to their unique genomic signature.

Comparative Analysis: HCT116 vs. HT29 Cell Lines

When juxtaposing HCT116 with HT29, another human colorectal carcinoma cell line, distinct differences in their oncogenic potential and differentiation capacities emerge:

Oncogenic Aggressiveness and Differentiation: HCT116 cells are characterized by their high oncogenic aggressiveness and limited differentiation potential, making them a model for studying aggressive tumor phenotypes. In contrast, HT29 cells exhibit the ability to differentiate into enterocyte-like and mucin-producing lineages, providing a contrastive model that mimics diverse aspects of colorectal cancer biology.

This comparative understanding of HCT116 and HT29 cell lines enriches the toolkit available to researchers, allowing for more nuanced investigations into colorectal cancer's multifaceted nature.

Pre-cancerous polyps growing in the colon.

Handling HCT116 cells

Doubling Time:

The doubling time for HCT116 cancer cells ranges from 25 to 35 hours.

Adherent or in Suspension:

The HCT116 colon cancer cell line is adherent, with cells growing in monolayers.

Seeding Density:

A seeding density of 2 x 104 cells/cm2 is recommended for the HCT116 cell culture. For subculturing, cells should be detached using Accutase solution after a 1x PBS wash. Following centrifugation, the cell pellet is resuspended in fresh growth medium and transferred to a new flask.

Growth Medium:

McCoys 5a medium, supplemented with 3.0 g/L L-glucose, 1.5 mM L-glutamine, 3.0 g/L NaHCO3, and 10% fetal bovine serum, is optimal for HCT116 cell cultivation. It is advisable to renew the medium 1 to 2 times per week.

Growth Conditions (Temperature, CO2):

Cultivation takes place in a humidified incubator at 37°C with a 5% CO2 atmosphere.


HCT116 cells can be stored at temperatures below -150°C in either the vapor or liquid phase of liquid nitrogen.

Freezing Process and Medium:

Use CM-1 or CM-ACF medium for cryopreservation. A controlled-rate freezing method is recommended, allowing for a gradual temperature decrease of 1°C per minute, which helps maintain cell viability.

Thawing Process:

Thaw HCT116 cells in a 37°C water bath. After adding growth medium, centrifuge to remove the freeze medium residues. Resuspend the cell pellet in fresh medium and culture in new flasks.

Biosafety Level:

Level 1

Cultured colon cancer cells HCT116 magnified at 20x and 10x.

HCT116 cell line: Advantages & Limitations

This section of the article will go over the advantages and limitations associated with the HCT116 cancer cell line.

Advantages of the HCT116 cell line

This section delves into the HCT116 cell line, highlighting its pivotal role in cancer research, particularly in the study of colorectal cancer, and discussing its inherent advantages.

Advantages of the HCT116 Cell Line

The HCT116 cell line stands out in cancer research due to several key advantages:

  • Colorectal Cancer Model: It serves as a widely recognized in vitro model for colorectal cancer, the third most common cancer globally. Its relevance to mimicking human colorectal cancer makes it invaluable for understanding cancer biology and testing therapeutic strategies.

  • Homogeneity: Remarkably, around 70% of HCT116 cells display consistent genetic profiles, offering a relatively homogeneous population. This uniformity is crucial for studies focused on gene expression, cellular signaling pathways, and assessing the efficacy of drug treatments, as it ensures consistency and reliability in experimental outcomes.

  • Transfection Efficiency: One of the distinguishing features of HCT116 cells is their high amenability to transfection, especially with viral vectors. This characteristic is particularly beneficial in gene therapy research, enabling the introduction of genetic material with efficiency and precision, thus facilitating advanced genetic manipulations and functional studies.

Advance your discoveries with our authenticated HCT116 cell line

Research applications of the HCT116 cell line

The HCT116 cell line has a wide range of applications in cancer research. Some prominent applications are:

Cancer biology

The HCT116 colon cancer cell line is used to study colon cancer progression and development. Moreover, it helps enlighten the underlying mechanisms and signaling pathways involved in cancer proliferation, migration, and invasion. A study used HCT116 cells to study genes involved in drug resistance development. Researchers overexpressed the MDR1 gene in colon cancer cells and observed the expression of NOX (NADPH oxide) isoforms and Nrf2. The study revealed that NOX2 and Nrf2 upregulation cause chemoresistance in cancer cells; thus, these genes can be targeted to overcome resistance development during cancer therapy [1]. Likewise, research conducted in 2021 reported that the NF-κB signaling pathway is involved in the regulation of colon cancer proliferation and migration. Thus, it can be targeted to develop new and effective therapeutics against colorectal carcinoma [2].

In the realm of oncology, understanding the intricate processes of cell cycle, proliferation and growth, and apoptosis is fundamental. These biological functions are pivotal in the study of human cell lines, particularly those derived from malignant cells such as human colon cancer cells and pancreatic cancer models. The HCT116 and SW620 cell lines, for instance, are instrumental in exploring the mechanisms underlying colon and pancreatic cancer, respectively. Through techniques like flow cytometry and clonogenic assays, researchers can elucidate the gene expression profiles and behavior of independent cells within tumors, shedding light on how cancer communicates within the extracellular matrix.

The Role of Apoptosis in Cancer Progression

Apoptosis, or programmed cell death, plays a critical role in maintaining cellular homeostasis and is a key area of study in cancer research. The distinction between unrelated apoptosis and apoptosis specifically induced within the context of cancer, such as colon cancer cell death, is crucial. This process is not just about the elimination of cells but involves a complex interplay of signals that can affect tumor growth and metastasis. By examining apoptosis and cell death in conjunction with metastasis suppressors and tumor suppressor activity, scientists can gain insights into the pathways that regulate cancer progression and metastatic potential.

Metastasis and Molecular Markers in Cancer

Metastasis remains one of the most formidable aspects of cancer, with hematogenous metastasis being a significant concern in the spread of malignant cells. The exploration of metastasis involves studying the movement and invasion capabilities of cancer cells, or cell locomotion, and how cells interact with their surroundings, including the extracellular matrix. Molecular markers like CD133 expression and the epidermal growth factor receptor are critical in identifying and understanding the behavior of positive colon carcinoma cells and other cancer types. The SIRT6 pathway, for example, has emerged as an area of interest due to its potential role in modulating tumor growth and metastatic colon cancer.

Toxicology/drug development

The HCT116 cell line is used as a screening model for new cancer drugs. Several studies have been conducted to assess the efficacy and toxicity of anticancer drugs, including natural products and chemically synthesized nanoparticles. As such, the research assessed the cytotoxicity of synthesized silver nanoparticles from the extracts of a herbal drug, Caesalpinia pulcherrima, in HCT116 cells [3]. In a study, researchers used the HCT116 cancer cell line to evaluate the anticancer potential of cocoa tea water extract. They found that cocoa tea extract reduces colon cancer proliferation and induces cell death [4]. Another study used HCT116 cancer cells and discovered that air potato, Dioscorea bulbifera, extracts exhibit pro-apoptotic activity in colorectal carcinoma cells via activation of the JNK signaling cascade and suppression of the ERK1/2 gene [5].

The effects of metformin on cancer cells, particularly in the context of colon and pancreatic cancer, exemplify how understanding the biological functions of cancer cells can lead to potential therapeutic strategies. Research into the clonogenic survival, or the ability to form clones, of cancer cells upon treatment with agents like metformin or targeting specific pathways such as the epidermal growth factor receptor can provide valuable insights into effective cancer treatments. Additionally, the use of HCT116 clones and HCT116 cell populations in these studies allows for a nuanced understanding of how cancer cells respond to different therapeutic interventions, paving the way for more personalized approaches to cancer treatment.

HCT116 cells: Research publications

This section will go over a few significant and most cited recent publications featuring the HCT116 cell line.

Cytotoxicity study of Piper nigrum seed mediated synthesized SnO2 nanoparticlestowards colorectal (HCT116) and lung cancer (A549) cell lines

This study was published in the Journal of Photochemistry and Photobiology B: Biology (2017). Researchers used HCT116 colon cancer and A549 lung cancer cell lines to evaluate the cytotoxic effects of piper nigrum seed-mediated synthesized tin oxide nanoparticles.

Long non-coding RNA SNHG15 interacts with and stabilizes transcription factor Slug and promotes colon cancer progression

This research in Cancer Letters (2018) proposes that lncRNA SNHG15 promotes colon cancer cell migration in colorectal cancer cell lines, including HCT116.

Overexpression of long non-coding RNA TUG1 promotes colon cancer progression

This paper was published in the Medical Science Monitor journal in 2016. The study found that oncogenic LncRNA TUG1 promotes the proliferation and migration of HCT116 colon cancer cells.

Drug resistance induces the upregulation of H2S-producing enzymes in HCT116 colon cancer cells

This research in the Biochemical Pharmacology journal (2018) proposes that drug resistance development upsurges the levels of H2S-producing enzymes in HCT116 colon cancer cells.

Apoptotic and antiproliferative effects of Inula viscosa L. water extract in the expression of microRnas on HCT 116 cell line: an in vitro study

This research paper in the International Journal of Environmental Health Research (2023) proposes that Inula viscosa L. extract exerts an anticancer effect on HCT116 colorectal cancer cells via regulating microRNAs.

Resources for HCT116 cells

Below are a few resources on HCT116 cells.

Frequently Asked Questions About HCT116 Cells

HCT116 is a cell line derived from human colorectal carcinoma, extensively utilized in cancer research to study the biology, genetics, and treatment responses of colorectal cancer.
In standard culture conditions, HCT116 cells, which exhibit an epithelial-like morphology, usually measure around 15 micrometers in diameter.
Although initially considered near-diploid, further genetic analyses have shown that HCT116 cells possess chromosomal abnormalities, rendering them aneuploid, which is a hallmark of many cancer cells.
The split ratio for HCT116 cells, which indicates the proportion of cells transferred to a new culture vessel during subculturing, typically ranges from 1:3 to 1:6, depending on the cell density and growth rate.
HCT116 and SW480 cells, both derived from colorectal carcinomas, differ in their genetic mutations, tumorigenic capabilities, and responses to drugs, reflecting the heterogeneity of colorectal cancer.
While both HCT116 and HT-29 cell lines are used in colorectal cancer research, they differ in their genetic makeup, morphological characteristics, and responses to chemotherapy, making each suitable for different research focuses.
HCT116 cells are favored in cancer research due to their well-characterized genetic background, reproducibility in experiments, and their relevance to human colorectal cancer, making them valuable for studies on cancer biology and drug efficacy.


  1. Waghela, B.N., F.U. Vaidya, and C. Pathak: Upregulation of NOX-2 and Nrf-2 promotes 5-fluorouracil resistance in human colon carcinoma (HCT-116) cells. Biochemistry (Moscow), 2021, 86, p. 262-274.
  2. Yang, M., et al., Astragalin inhibits the proliferation and migration of human colon cancer HCT116 cells by regulating the NF-κB signaling pathway. Frontiers in Pharmacology, 2021, 12: p. 639256.
  3. Deepika, S., C.I. Selvaraj, and S.M. Roopan, Screening bioactivities of Caesalpinia pulcherrima L. swartz and cytotoxicity of extract synthesized silver nanoparticles on the HCT116 cell line. Materials Science and Engineering, C, 2020, 106, p. 110279.
  4. Gao, X., et al., Cocoa tea (Camellia ptilophylla) induces mitochondria-dependent apoptosis in HCT116 cells via ROS generation and the PI3K/Akt signaling pathway. Food Research International, 2020, 129, p. 108854.
  5. Hidayat, A.F.A., et al., Dioscorea bulbifera induced apoptosis through inhibition of ERK 1/2 and activation of JNK signaling pathways in HCT116 human colorectal carcinoma cells. Biomedicine & Pharmacotherapy, 2018. 104: p. 806-816.


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