Engineering strategies to capture the biological and biophysical tumor microenvironment in vitro

Abstract

Despite decades of research and advancements in diagnostic and treatment modalities, cancer remains a major global healthcare challenge. This is due in part to a lack of model systems that allow investigating the mechanisms underlying tumor development, progression, and therapy resistance under relevant conditions in vitro. Tumor cell interactions with their surroundings influence all stages of tumorigenesis and are shaped by both biological and biophysical cues including cell-cell and cell-extracellular matrix (ECM) interactions, tissue architecture and mechanics, and mass transport. Engineered tumor models provide promising platforms to elucidate the individual and combined contributions of these cues to tumor malignancy under controlled and physiologically relevant conditions. This review will summarize current knowledge of the biological and biophysical microenvironmental cues that influence tumor development and progression, present examples of in vitro model systems that are presently used to study these interactions and highlight advancements in tumor engineering approaches to further improve these technologies.

Keywords: Cancer heterogeneity; Cancer metabolic transport; Extracellular matrix; Tumor engineering; Tumor microenvironment; Tumor on a chip.

Figure 1:. Biological and biophysical hallmarks of the tumor microenvironment.

Schematic of the cellular (cancer-associated fibroblasts [CAFs], endothelial cells, and immune cells) and acellular features (fibrotic remodeling of the extracellular matrix [ECM], metabolic/cytokine gradients, and interstitial fluid flow and pressure) of the tumor microenvironment. Increased recruitment of CAFs leads to compositional, structural, and mechanical changes of the ECM, while vascular dysfunction impacts mass transport of oxygen, nutrients, and paracrine signaling molecules. The latter results in varied gradient formation of solutes, elevated interstitial pressure and fluid flow as well as central necrosis. Collectively, these changes promote tumor progression and invasion where cancer cells evade immune surveillance, breach the basement membrane, migrate into the surrounding stroma, and eventually intravasate into an adjacent blood vessel to metastasize to distant organs. Image created with BioRender.com.

Figure 2:. Multicellular spheroid and organoid models.

A. Multicellular spheroids formed from a single cancer cell line recapitulate diffusion limited transport resulting in spatially distinct regions of proliferation and necrosis that mimic the pathophysiological heterogeneity of tumors in vivo. Reproduced from Impact Journals, LLC with an open-access copyright policy. [134]. B. Co-culture spheroids containing tumor cells (red), CAFs, and monocytes recapitulate stromal heterogeneity. Cisplatin treatment caused rapid apoptosis (green) in these cultures, while paclitaxel was less effective. Reproduced from Elsevier Ltd with an open-access copyright policy. [136]. C. Mouse pancreatic stellate cells (PSCs, red) and pancreatic cancer organoids (green) can be embedded in Matrigel to increase stromal populations. PSCs cultured in Matrigel alone remain quiescent, but PSCs in contact with tumor organoids assume CAF-like phenotypes. Reproduced with permission from Rockefeller University Press [139]. D. Different types of patient-derived tumor organoids (bottom row) can be formed via the air–liquid interface (ALI) method, a technique that recapitulates parental tumor histology (top row) and maintains immune cell populations. Reproduced with permission from Elsevier Ltd [143].

Figure 3:. Biomaterials models to study cell-ECM interactions.

A. Culture in Matrigel® can be used to test the effect of malignant transformation on morphogenesis. Normal epithelial cells (MCF10A) form small acinar structures similar to human mammary tissues (top right) when cultured in Matrigel®, while transformed cells (human papillomavirus E7 [HPV E7] and erythroblastic oncogene B [ErbB2]) are larger and compose multiacinar structures. Reproduced with permission from Elsevier Ltd. [156]. B. (i) When embedded into collagen, co-culture spheroids composed of tumor cells and obesity-associated stromal cells invade collectively due to collagen displacement, a process that can be partly inhibited by MMP inhibition with Batimastat. Reproduced with permission from John Wiley & Sons, Inc. [135]. (ii) Culturing breast cancer cells in aligned collagen fiber matrices induced morphological changes (top images, cells in green, collagen in grey) that led to increased directional migration (bottom image, cells in cyan). Reproduced with permission from Elsevier [178]. C. Decellularized ECM can be derived from fibroblasts cultured in vitro. ECM derived from alpha-SMA (red) positive, desmoplastic human fibroblasts is more aligned and enriched for fibronectin (green) compared to ECM derived from normal fibroblasts. Reproduced with permission from Elsevier [182]. D. A hybrid alginate hydrogel modified with PEG spacers enables introduction of varied viscoelastic properties. Faster stress relaxation (left to right) promoted osteogenic differentiation of mesenchymal stem cells as indicated by increased matrix mineralization (Von Kossa staining, top panel) and type-1 collagen deposition (bottom panel). Reproduced with permission from Springer Nature [203]. E. Electrospun networks of RGD-modified dextran methacrylate (DexMA) fibers permit independent tuning of fiber and bulk mechanical properties and revealed that soft fiber stiffness (top panel) promotes cell spreading (cells outlined in magenta) typically associated with stiff matrices by enabling cellular recruitment of individual fibers. Reproduced with permission from Springer Nature [215].

Figure 4:. Microfabricated models to study transport considerations.

A. Schematic demonstrating transport considerations in the tumor microenvironment, including gradients of secreted factors, nutrients and oxygen, and elevated interstitial flow and pressure. Microfabricated in vitro models can isolate these aspects to determine their contribution to tumorigenesis. Image was created with BioRender.com. B. A TRACER model consisting of a sheet of cells rolled onto an oxygen impermeable membrane creates a naturally derived oxygen and nutrient gradient enabling spatial mapping of cell metabolism and phenotype. Reproduced with permission from Springer Nature [228]. C. An in vitro 3D culture model consisting of a Boyden chamber containing a cell-laden hydrogel was used to mimic the effects of interstitial flow in conjunction with chemokine signaling on tumor cell migration through a 3D matrix. Reproduced with permission from Elsevier [237]. D. A PDMS-based microfluidic vascularized model composed of an endothelial cell-coated channel (green), a central hydrogel channel, and a tumor cell channel (red) was used to investigate how endothelial barrier function influences tumor cell intravasation. Reproduced with permission from the National Academy of Sciences [244]. E. Endothelial cell-coated microfluidic channels with and without pericyte stabilization can also be formed directly within a collagen bulk preventing cell-contact with PDMS surfaces that may cause artefacts. Reproduced with permission from Springer Nature [252].

Figure 5:. Organ/body-on-a-chip systems and 3d printed models.

A. Organ-on-a-chip models have been used to study bone-specific metastatic colonization by microfluidic integration of mineralized bone matrix, bone marrow stem cells, and endothelial cells. This device also enabled interstitial flow generation, revealing that flow increased cancer cell proliferation. Reproduced with permission from The National Academy of Sciences [269]. B. By integrating multiple organ-on-a-chip systems, a body-on-a-chip platform was used to measure anti-tumor efficacy and cardiotoxicity, demonstrating that the treatment responses of Ewing Sarcoma tumors and heart muscle to Linsitinib was closer to clinical trial results in this integrated system compared to treatment in isolated tissues. Reproduced with permission from The Royal Society of Chemistry [273]. C. Additive 3D bioprinting enables the fabrication of tissue scale constructs with complicated structures in conjunction with a temperature sensitive hydrogel support bath. The feasibility of this approach was demonstrated by printing, (i) a heart valve and (ii) a subregion of vascularized cardiac tissue. Reproduced with permission from The American Association for the Advancement of Science [282]. D. Negative-space bioprinting involves casting a material such as agarose around a 3D printed sacrificial template, which can generate complex architectures, for example, in vascular beds. Reproduced with permission from Springer Nature [287].

Figure 6:. Opportunities to improve engineered tumor models.

The individual tumor models presented in this review vary in their complexity and span a range of engineering approaches to fabricate them. Integrating multiple of these different models and approaches within one integrated system represents an opportunity to develop the next generation of tumor models. Additional opportunities can be derived from integrating technologies from other disciplines such as imaging, advanced omics, and computational approaches as well as clinical/patient-oriented aspects such as using these models for cancer drug screening in precision medicine settings and improving scale-up manufacturing. Image was created with BioRender.com.