Most cancer deaths are due to tumor metastasis rather than the primary tumor. Metastasis is a highly complex and dynamic process that requires the orchestration of signaling between the tumor, its local environment, distant tissue sites, and the immune system. Animal models of cancer metastasis provide the necessary systemic environment but lack control over factors that regulate cancer progression and often do not recapitulate the properties of human cancers. Bioengineered “organs-on-a-chip” that incorporate the primary tumor, metastatic tissue targets and microfluidic perfusion are now emerging as quantitative human models of tumor metastasis. The ability of these systems to model tumor metastasis in individualized, patient-specific settings makes them uniquely suitable for studies of cancer biology and developmental testing of new treatments. In this review, they focus on human multi-organ platforms that incorporate circulating and tissue-resident immune cells in studies of tumor metastasis.
Despite enormous advances in their understanding of tumor progression, cancer incidence, mortality, and cost of treatment continue to increase. Recent breakthroughs in cancer modeling and therapy have been primarily directed at early intervention and eradication of the primary tumor, which has limited success in the presence of metastatic disease. Although tumor metastasis accounts for 66.7% of the mortality related to solid tumors, few drugs are designed to specifically target metastatic spreading. This unmet need is due, at least in part, to the lack of predictive human models that recapitulate physiological metastasis and can be used for mechanistic studies of metastatic progression and evaluation of drug candidates that can prevent or treat metastasis in patients.
Metastasis develops as cancer cells are leaving the primary cancer site to find a more suitable niche in the body. This process involves cascades of multidirectional signaling between tumor cells, stromal cells, and immune cells within the primary and secondary tumor sites. Tumor cells co-opt the immune system, enabling escape from tumoricidal immune responses, and alter the primary tumor environment (TME) to promote vascularization. Collectively, these changes facilitate tumor cell invasion through the stroma and migration to nearby vessels, the first step of the metastatic cascade.
A) The progression from primary to secondary tumor sites involves (1) remodeling of the primary tumor microenvironment to support (2) intravasation of circulating cancer cells into vascular circulation. Steps (1) and (2) work in conjunction with step (3) inter-organ communication between the primary tumor, bone marrow, and immune organs, to prime the pre-metastatic niche in distant target tissues. The pre-metastatic niche facilitates (4) extravasation of cancer cells from the circulation into a tissue bed, and (5) targeted cell engraftment in the secondary metastatic site.
(B) Bioengineered systems can be leveraged to deconstruct each of the steps in metastatic progression and improve their understanding of the dynamic interplay between the primary tumor, targeted tissue sites, and immune cells and organs.
Tumor cells then intravasate, via blood or lymphatic vessels, into the circulation, and survive as they traffic to microvessels at distant, metastatic sites that have been primed for colonization. Arrested tumor cells subsequently extravasate into the tissue and proliferate to form a secondary tumor. Two theories have been proposed to explain organ-specific homing and selection of metastatic sites: (1) Paget's “seed and soil” theory, first described in 1889, which posits that circulating cancer cells preferentially “seed” distal organs where the microenvironment (“soil”) favors their survival, and (2) Ewing's “flow and filter” theory, described in 1929, which posits that circulating tumor cells are mechanically driven to metastatic sites by blood flow patterns that are governed by capillary bed sizes and adhesion. Recent work has demonstrated contributions of both mechanisms to the establishment of metastatic sites.
For decades, cancer research has relied on the use of monolayer cultures of cancer cells for studies of cancer biology and drug screening, followed by the use of genetically engineered mice or patient-derived xenotransplants (PdX) for a better understanding of the systemic aspects of the disease. The development of engineered cancer models has been motivated by the need for re-creating in vitro many aspects of the disease that are otherwise missed in these established in vitro and in vivo models. While some of the oldest 3D tumor models date back to more than 30 years, there have been major advances in introducing bioengineering methodologies into the field of cancer, toward developing increasingly accurate, useful, practical, and complex models for studying metastasis.
Traditional models to study primary tumors and resected secondary metastatic cells include 2D cell monolayers and animal models, where cells are either implanted in vivo into an immunodeficient mouse or a transgenic mouse line is created with a known mutational defect. Bioengineered models include 3D multi-cellular tissue-engineered models, patient-derived organoids, organ-on-a-chip microfluidic devices, and recently, multi-organ-on-a-chip devices. They show here the overview of each approach and its advantages and limitations in studying cancer.
Generally speaking, four approaches are currently utilized to model human cancer: (1) cancer cell culture in 3D scaffolds, (2) patient-derived organoids, (3) single organ-on-a-chip systems, and most recently, (4) multi-organ-on-a-chip systems. Among these approaches, the growth of cancer cells as self-assembled spheroids within hydrogels and scaffolds is most commonly used. However, given that metastasis is a dynamic process that involves multiple tissues and relies on the systemic circulation for its spread, microfluidic single- and multi-tissue OOC platforms have been proved to be most useful for studying metastatic progression.
Cancer cell culture in 3D scaffolds
Arguably the most widely published approach using cancer cell lines has been to grow them in natural and synthetic scaffolds providing a regulatory 3D setting for tumor cells. Complex techniques have since been developed that enable the generation of scaffolds with distinct compositions, structures, and mechanical properties. However, the static culture of cancer cells in a 3D microenvironment is inherently limited in its ability to model the multiple stages of metastasis. Several recent reviews on engineering the 3D microenvironment have been provided by others.
Although practical for laboratory research, cancer cell lines fail to recapitulate the heterogeneity of the primary cancer cell populations. The development of patient-derived tumor organoids representing a wide variety of primary cancer types has overcome this limitation. Critically, these tumor organoids have been shown to preserve inter-patient genetic diversity, both for the primary and metastatic tumor sites. They have also been proved to be successful at modeling previously difficult to study cancer types, such as pancreatic and prostate cancers. CRISPR-Cas9-mediated genetic engineering of healthy epithelial tissue organoids has allowed for the investigation of driver oncogenes and tumor suppressors in tumorigenesis. Perhaps of most relevance to metastasis and improving the poor clinical outcomes associated with it, the development of patient-derived organoids has the potential for the personalization of cancer treatment. For this reason, there has been wide interest in the use of tumor organoid models for drug sensitivity testing. Unfortunately, most advances in the organoid field were with carcinomas (epithelial cell-derived cancers), given their intrinsic ability to self-assemble into micro-tissues in culture. This concept of using patient-derived materials has begun to be extended to modeling other cancer types. One such case involved multiple myeloma, where bone marrow aspirates were taken from patients and reconstituted in co-cultures of patient cancer cells and stromal cells within patient bone marrow ECM hydrogels.
Single organ-on-a-chip models
Static cultures of cancer cells, whether in scaffolds or as organoids, prevent the incorporation of biophysical stimuli that cancer cells experience in the body. The microfluidic-based OOC systems incorporate fluid flow, to recapitulate metastatic cell circulation and activate the associated biomechanical regulation pathways. For example, the physiologically relevant fluid flow in a model of the human bone perivascular niche allowed for metastatic breast cancer colonization, with a recapitulation of shear stress-induced increases in metastatic resistance of breast cancer cells.
The use of microfluidic OOCs has also enabled investigations into the interactions of cancer cells with the endothelium, which are known to mediate tumor survival and metastatic progression. Perfusion allows re-creating in vitro stable and functional vascular networks that are capable of supporting micro-tumors. Microfluidic models have additionally empowered the ex vivo studies of clinically relevant intravasation and metastasis. Remarkably realistic capillary networks can be formed to study metastatic cell intravasation with high precision, as single cells have been visualized crossing engineered endothelial barriers.
The downstream processes involved in extravasation have also been modeled using microfluidic OOCs for metastatic breast cancer cell circulation and extravasation into bone, a common secondary tumor site. Recent advances in these approaches have even allowed for the evaluation of metastatic propensity of patient-derived cancer cells, as well as for the prediction of treatment efficacies of drugs against tumors formed at metastatic sites. Finally, OOCs have been proved to be valuable for drug screening through their ability to re-create biological mass transport of drugs experienced by tumors adjacent to capillaries, and thereby allow studies of the regulatory roles of microvascular endothelial cells.
As the capabilities of microfluidic OOCs and the confidence in the predictability of these systems have increased, so has their complexity, with several groups creating interconnected multi-tissue platforms. The incorporation of several tissue systems allows for studies of systemic effects, such as organ-specific homing and metastasis. Other advances have been motivated by the need to improve preclinical evaluation of anticancer drugs, which are notorious for the low predictability of both drug toxicity and efficacy. The expectation is that engineered malignant and healthy tissues can better recapitulate human physiology and lead to a more accurate preclinical evaluation of candidate therapeutics. Using multi-tissue systems allows physiologically relevant ways of drug testing and measurements of both “target” effects (on tumor tissue) and “off-target” toxicity (on healthy tissues) This approach has shown promise in identifying clinically relevant resistances to experimental small molecule treatments and has been used to demonstrate metabolism of such drugs, and the subsequent cardiotoxic effects of the metabolites within an in vitro context1.
Pamela L. Graney, Daniel Naveed Tavakol, Alan Chramiec, Kacey Ronaldson-Bouchard, Gordana Vunjak-Novakovic,
Engineered models of tumor metastasis with immune cell contributions,
Volume 24, Issue 3,