Transgenic mouse model

The ‘Onco-quest’: Decoding Breast Cancer Modelling

Picture a remarkable scientific voyage that carries us from observing cells in a laboratory dish to crafting lifelike replicas of human organs, resembling miniature worlds within the palm of our hands. It’s like a transition from mere sketches to an awe-inspiring 3D masterpiece. This journey, similar to the artistry of creating captivating models, resonates far beyond the boundaries of art alone.

Forecasting and planning with reliable models is a valuable asset across various sectors, driving efficiency, innovation, and overall progress. Disease modelling plays a crucial role in improving healthcare outcomes. It involves creating lab-based biological systems that mimic diseases or showcase the underlying disease processes. From disease diagnosis to drug discovery, from patient monitoring to optimising healthcare operations, the utilisation of reliable disease models stands evident.

With its devastating toll on lives and spurring a worldwide quest for answers, cancer remains an intense focus of research. Without a definitive cure, scientists worldwide are propelled to pioneer innovative models for studying cancer physiology and crafting effective treatments. Among the diverse forms of this disease, breast cancer stands as the second leading cause of death among women. In the light of its gravity, disease modelling techniques have undergone a continuous evolution. From traditional cell line cultures, researchers have ventured into the realm of 3D cell culture models, and now, they forge ahead in advancing artificial organoids, marking a significant leap forward.

Cell culture development significantly changed the area of life sciences, providing a foundation for our initial understanding of cancer biology. Primary cancer cell lines are ex vivo cell populations deriving directly from resected tissue samples, most commonly from core biopsies, pleural effusion, or autopsy specimens. These in-vitro model systems helped us study cancer’s cellular behaviour and the underlying genetic and epigenetic alterations that drive tumour initiation and progression.

In the context of breast cancer, the prominent cell lines used are MCF-7, T47D, and MDA-MB-23. Trastuzumab, a drug formulated through the efforts of breast cancer cell cultures, stands as a testament to the immense potential of these cell lines. Yet, as the wheels of progress turned, the limitations of traditional cell culturing methods became apparent. Tumour cell lines acquire mutations during the culture process, which cannot faithfully simulate the original characteristics of the tumour. In addition, cell cultures cannot mimic the interactions between tumour cells and other stromal cells in vivo, as cultured cells are single and lack the hierarchy of different cell types.

As the pursuit of knowledge advanced, the spotlight shifted to xenotransplantation studies, serving as the backbone of oncology research for over four decades. A xenograft is a piece of living tissue taken from a donor of one species and grafted into a recipient of different species. Breast cancer tissue xenotransplantation involves implanting small fragments of breast tumour tissue from cultivated cell lines into murine models, often into their mammary fat pad (orthotopic) or under the skin (subcutaneous). Within these hosts, tumours take root, grow and develop, which is monitored over time. The important aspects of tumour studies known using this are measuring tumour volume, monitoring tumour growth kinetics, and assessing tumour histology.

Moving one step ahead into the zone of personalised treatments, these xenograft models evolved into what we know as the patient-derived xenografts (PDX) models. These include deriving patient tumour samples and transplanting them into immunodeficient mice. These models retain the characteristics and heterogeneity of the original tumour and are used to study individual patient responses to therapies, drug resistance, and tumour evolution. Yet, like any successful journey, challenges lie in wait. Variations in the tumour microenvironment and differences in immune interactions between humans and mice influence therapy response. Additionally, the time-consuming long culture cycles of the host animal make it inefficient and difficult for high-throughput drug screening work.

Mice serve as valuable models for cancer research due to their shared genomic and physiological features with humans, enabling the study of fundamental cancer mechanisms and treatment responses. With 80% of mouse genes having human counterparts, it is an excellent experimentally tractable model system.

Traditionally, researchers used immunocompetent and immunodeficient mice with subcutaneously or orthotopically transplanted xenografted tumours. In a groundbreaking experiment in 1974, R. Jaenisch and B. Mintz injected viral oncogenes from the simian virus (SV40) into mouse embryos. Although the resulting mice did not develop tumours, they revealed the presence of integrated viral DNA in cells across various tissues. These mice were the pioneers of transgenic models.

Taking it a step further, in 1984, Harvard researchers Philip Leder and Timothy Stewart accomplished a remarkable feat: creating transgenic mice specifically designed to study breast cancer. A retrovirus, called MMTV (mouse mammary tumour virus), was known to cause mammary tumours in certain strains of mice, and its regulatory region had been mapped. By isolating the key DNA sequence from the virus and combining it with cancer-promoting oncogene myc, Leder and Stewart constructed a hybrid DNA sequence. By microinjecting this sequence into mouse embryos, they successfully developed the MMTV-myc mouse model, Oncomouse. The first patented animal, Oncomouse, could develop inheritable breast tumours, offering an invaluable tool for studying the intricacies of cancer.

With synthetic biology’s foray into the modern world, we witnessed further complex techniques in the field of genetic engineering. The availability of the complete sequence of the mouse genome, the technology to manipulate it, and well-defined inbred strains, made it possible to engineer mice to test the hypotheses of tumorigenesis. Experiments can now be easily undertaken to assess the outcome when the function of a gene is lost, mutated, underexpressed, or overexpressed in the appropriate cell types in vivo. Genetically engineered mouse models took a giant leap with the latest discovery of the CRISPR-CAS 9 technology.

CRISPR-Cas9 is an RNA-guided nuclease involved in adaptive immunity in bacteria and archaea. Cas9 is guided by programmable RNA known as the single guide RNA (sgRNA). The Cas9/sgRNA complex recognizes the complementary genomic sequence and makes cuts to induce double-strand DNA breaks. These breaks are then repaired by the cell’s DNA damage repair mechanisms which gives rise to small insertions and deletions in the DNA leading to precise DNA modification.

CRISPR-Cas9 genome editing has made it easy, fast, and effective to build precision cancer models. It has already been used to generate knockout or knock-in mouse models for precision cancer studies.

1. Studying Loss of Gene Function in Mouse Models

In knockout mouse models, a specific gene of interest is intentionally inactivated or “knocked out”. This is typically achieved by disrupting the gene’s DNA sequence, preventing its normal expression and function. For breast cancer studies, researchers have targeted genes such as BRCA1 and BRCA2, known as the tumour suppressor genes. These genes help maintain genomic stability and prevent the formation of cancerous cells. By creating knockout mice lacking BRCA1 or BRCA2, scientists investigate the consequences of gene loss and better understand the mechanisms underlying breast cancer development.

2. Studying Gain of Gene Function in Mouse Models

Knock-in mouse models involve introducing specific genetic alterations or modifications into the genome. This can be achieved by inserting mutated versions of genes, known as oncogenes, into the mouse genome. In breast cancer studies, researchers have utilised knock-in models to study genes such as HER2/neu (also known as ERBB2), which is frequently amplified and overexpressed in a subset of breast cancers. By introducing an activated form of the HER2/neu gene into mouse models, scientists can observe the effects of its overexpression on breast tumour formation and progression.

Over the years, researchers have diligently explored various factors associated with breast cancer, yet many drug-testing models have failed in pre-clinical trials. Breast cancer is a highly complex disease, influenced by genetic, environmental, and lifestyle factors, and displays significant variability in tumorigenesis even within patients sharing the same cancer type. To tackle this individual variability, personalised treatment approaches have emerged, tailoring therapies to meet each patient’s unique needs.

One remarkable advancement in this field is the creation of organoids: lifelike replicas of human organs grown in a laboratory setting. These three-dimensional cultures provide a valuable tool for disease modelling, including breast cancer. Organoids can be easily manipulated, enabling detailed study of cell-to-cell and cell-to-extracellular matrix interactions. Recent studies have demonstrated that breast tumour organoids cultivated in 3D culture accurately represent the diverse characteristics found in different breast cancer subtypes, making them particularly valuable for investigating the effects of therapeutic interventions.

In the context of breast cancer, the healthcare industry has successfully created breast organoids. Initially, patient-derived breast epithelial subsets are isolated and purified to culture-specific cell lines. These cells are then embedded in an extracellular matrix and cultured in a specialised medium containing growth factors. Over time, the cells proliferate and differentiate, forming three-dimensional structures that closely resemble breast tissue. As the organoids mature, they acquire structural and functional characteristics similar to the breast.

Taking the research further, scientists employed CRISPR-Cas9-mediated gene editing in human breast organoids to mimic neoplasia, the abnormal growth of cells that leads to cancer. They selected P53, PTEN, and RB1 — three tumour suppressor genes frequently mutated in breast cancer for editing. Cas9 was introduced into normal organoids, followed by the sequential introduction of lentiviruses expressing single-guide RNAs targeting P53, PTEN, RB1, or control single-guide RNAs.

Organoid culture technology can be used for high-throughput screening of antitumor drugs. In addition, organoid culture maintains the original genotype and biological characteristics of the tumour, thereby facilitating the design of ultra-precision individualised treatment strategies.

Although organoids have broad application prospects, they lack mesenchymal cell support, nerve innervation, and vascular support. Therefore, there is still a considerable gap between organoids and real organs. The prospects include bridging the gaps between organoids and real organs by incorporating multiple cell types in organoid models.

The dynamic journey of modelling from the early 80s to the present day is an instructive example of the unfolding of scientific discovery in biology, where findings rarely come unilaterally but evolve out of a melting pot of ideas, results, failures, and unexpected outcomes. While each modelling approach has experienced its fair share of triumphs and setbacks, they have played a pivotal role in unravelling the complexities of oncology studies and advancing drug development. The global chase for a cure to mitigate the devastating impact of cancer remains, and the continual innovation in modelling techniques serves as a guiding light for subsequent steps in cancer treatment — ranging from early cancer detection to drug development, testing, and delivery. Together, these advancements propel us closer to the ultimate goal of conquering this disease.

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