Orthotopic and Metastatic Tumor Models for Preclinical Research

Pharmacology & Therapeutics. 2024 May; 257: 108631.

Authors: Stribbling SM, Beach C, Ryan AJ.

While significant progress has been made in cancer treatment over recent decades, novel approaches to the development of new drugs remain crucial to address continuing unmet clinical needs. Orthotopic and metastatic tumor models allow drugs to be evaluated in more clinically relevant biological contexts. By considering target expression, drug distribution, and the interactions between tumor cells, non-tumor stromal components, blood vessels, and the host immune system, these models provide a more comprehensive assessment of treatment responses. Data generated in orthotopic models may more closely resemble the clinical disease and may be a more accurate guide to biomarker development and decision-making in clinical trials thereby improving the predictive value of preclinical findings. However, the use of orthotopic and metastatic tumor models for drug evaluation poses some practical difficulties. For example, the surgical procedures, post-operative care, and monitoring of tumor growth at specific anatomical sites all add complexity and can increase overall experimental timelines. Additionally, orthotopic tumor models generally have higher experimental variability compared to subcutaneous models meaning larger group sizes may be required to reliably assess drug effects.

Orthotopic Tumor Models

Orthotopic tumor models aim to replicate the clinical setting of tumor growth and progression by implanting tumor cells or tissues at their anatomically correct or appropriate location within the body of an experimental animal. A goal of orthotopic tumor models is to recreate the natural microenvironment of the tumor site, including tissue-specific architecture, cell-cell interactions, and vasculature, to better mimic the complex interactions and behavior of tumors in the human body.

Many anatomical sites have been used to set up orthotopic tumor models in mice reflecting the wide variety of human cancers being studied. As well as the four most commonly diagnosed cancers - breast, prostate, lung, and colon - there are significant proportions of patients with newly diagnosed cancers at other anatomical sites including the pancreas, bladder, ovary, and liver, and each anatomical site represents an important disease for orthotopic cancer models.

• Human tumor cell-line derived xenografts (CDXs) (from "xeno-" foreign; graft from one species to an unlike species) are the most widely used subcutaneous tumor model and they are also a widely used orthotopic tumor model system. For xenograft models such as these, tumor cells/fragments are injected/implanted into immune-deficient mice, many strains of which are now available. In contrast, murine tumor cell lines can be grown in genetically matched (syngeneic) fully immune-competent mice, using widely available inbred strains such as C57BL/6 and BALB/c.
• The problem can be overcome to a certain extent by using patient-derived xenograft (PDX) models. Fresh tumor tissue derived from treatment-naïve primary or metastatic tumors is obtained during surgery or from biopsies (or occasionally, from ascites). Typically, the tumor is cut into small (1-2 mm³) pieces or disaggregated enzymatically or mechanically to give a cell suspension, and then engrafted or injected ectopically into immune-deficient mice, with several potential sites being available, including subcutaneously in the flank, in the anterior compartment of the eye, under the renal capsule, or into the intracapsular fat pad. More recently, PDX models have also been established from patient-derived circulating tumor cells (CTCs) rather than the tumor itself. PDX tumors can also be engrafted orthotopically into the same organ as the original tumor and grown as patient-derived orthotopic xenografts (PDOXs). PDX and PDOX tumors maintain much of the structure and composition of the parent tumor and more accurately recapitulate the human disease. As a result, the response of patients to certain types of therapy may be more accurately predicted. Importantly, PDOX models better mimic clinical metastases than subcutaneous PDX models, suggesting they have greater biological and clinical relevance.

Tumor Organoid Models

An organoid can be defined as a 3D structure grown from stem cells and consisting of organ-specific cell types that self-organize through cell sorting and spatially restricted lineage commitment. There has been growing interest in extending the concept of tissue organoids to encompass the use of patient-derived tumor organoids to create in vivo models. To establish organoids, tumor cells can be derived from patient biopsies, surgically resected tumors, PDX tumors, or genetically engineered mouse models. Tumor cells are embedded in an extracellular matrix or scaffold that provides a three-dimensional structure for growth. Specific growth factors and nutrients are added to support the growth and survival of tumor cells and, over time, the tumor cells in 3D culture self-organize into organoids, forming tissue-like structures mimicking the cellular heterogeneity seen in the original tumor with expression of cell-specific markers and the development of differentiation-associated properties such as secretory functions in glandular tumors. Patient-derived tumor organoids have been established for a broad range of tumor types and have several practical advantages over patient-derived xenografts including a higher establishment success rate, more rapid establishment, and the potential to generate matched normal control tissue. Cancer cells can be converted between organoid culture and xenografts with high efficiency with the result that patient-derived organoids have certain benefits of both 2-D cultured cells (e.g. ease of growth, genetic manipulation, implantation success) and PDXs (e.g. orthotopic implantation and metastasis, disease-relevant tumor microenvironment/stroma).

Organoids have some potential limitations, such as decreased cell diversity and heterogeneity compared with PDXs and lack of accepted standardized methods for culture/propagation but the benefits of these cells for establishing orthotopic/metastatic models and the ongoing development of disease-specific organoid biobanks suggest that they will find increased use in the future across a broader range of tumor types, including rare cancers.

Humanized Mice

Human CDX, PDX, and organoid orthotopic models require the use of immune-deficient mice with strains that exhibit severe immune deficiencies such as the NOD-SCID-IL2R gamma null (NSG) strain which is most often used for engraftment of primary human samples. The lack of an intact immune system is a significant limitation of orthotopic models grown in immune-deficient mice, but more recently NSG mice in particular have been used to establish humanized mouse models, where the defective mouse immune system is replaced ("humanized") with e.g. human peripheral blood mononuclear cells (PBMCs) or hematopoietic stem cells (HSCs) to recapitulate the human immune system. This approach has enabled the role of the immune system in cancer therapy to be investigated using xenografted human tumors rather than syngeneic mouse tumor models. Although humanized mice have several advantages, there are several potential limitations: they do not fully recapitulate the human immune system; the human-derived PBMCs or HSCs are not patient-matched to the tumor; and, the development of graft-versus-host disease due to the interaction of the humanized immune system with mouse tissues may occur. Approaches to address some of these shortcomings are being investigated. For example, an immune-deficient mouse model has been developed that expresses human HLA instead of mouse MHC where the immune deficiency can be corrected by transferring functional HLA-matched PBMCs resulting in an immune-competent mouse with a more human-like immune system.

Metastatic Models

The transport of tumor cells from the primary to distant sites occurs mainly via the circulatory and lymphatic systems, in theory enabling viable circulating tumor cells (CTCs) to spread and colonies almost any tissue in the body. However, in practice, for many primary tumors, the potential anatomical sites of secondary tumor growth are more restricted. Predating our current understanding, Paget in 1889 suggested that the outcome of metastasis depends upon interactions between the tumor cells and the host tissue, with the metastatic tumor cell ("seed") being able to grow into a secondary tumor only once it has reached a sustaining organ environment ("soil"). Today, we can think of the seed in terms of e.g. cancer stem cells, progenitor cells, or initiating cells, whereas the soil encompasses specific stromal and microenvironmental factors that together constitute an amenable pre-metastatic niche. In consequence, the site of formation of metastases can depend on specific interactions between the CTCs and the prospective host growth site leading to specific patterns of metastatic spread for specific cancer types, with the main organ sites of metastasis for the commonest cancers being the liver, lung, bone and brain.

Creative Bioarray Relevant Recommendations

Disease Models

Reference

1. Stribbling SM, et al. (2024). "Orthotopic and metastatic tumor models in preclinical cancer research." Pharmacol Ther. 257: 108631.

For research use only. Not for any other purpose.