αCT1 peptide sensitizes glioma cells to temozolomide in a glioblastoma organoid platform.

Glioblastoma (GBM) is the most common form of brain cancer. Even with aggressive treatment, tumor recurrence is almost universal and patient prognosis is poor because many GBM cell subpopulations, especially the mesenchymal and glioma stem cell populations, are resistant to temozolomide (TMZ), the most commonly used chemotherapeutic in GBM. For this reason, there is an urgent need for the development of new therapies that can more effectively treat GBM. Several recent studies have indicated that high expression of connexin 43 (Cx43) in GBM is associated with poor patient outcomes. It has been hypothesized that inhibition of the Cx43 hemichannels could prevent TMZ efflux and sensitize otherwise resistance cells to the treatment. In this study, we use a three-dimensional organoid model of GBM to demonstrate that combinatorial treatment with TMZ and αCT1, a Cx43 mimetic peptide, significantly improves treatment efficacy in certain populations of GBM. Confocal imaging was used to visualize changes in Cx43 expression in response to combinatorial treatment. These results indicate that Cx43 inhibition should be pursued further as an improved treatment for GBM.

Patient-Specific Sarcoma Organoids for Personalized Translational Research: Unification of the Operating Room with Rare Cancer Research and Clinical Implications.

INTRODUCTION: Sarcoma clinical outcomes have been stagnant for decades due to heterogeneity of primaries, lack of comprehensive preclinical models, and rarity of disease. We hypothesized that engineering hydrogel-based sarcoma organoids directly from the patient without xenogeneic extracellular matrices (ECMs) or growth factors is routinely feasible and allows rare tumors to remain viable as avatars for personalized research. METHODS: Surgically resected sarcomas (angiosarcomas, leiomyosarcoma, gastrointestinal stromal tumor, liposarcoma, myxofibrosarcoma, dermatofibrosarcoma protuberans [DFSP], and pleiomorphic abdominal sarcoma) were dissociated and incorporated into a hyaluronic acid and collagen-based ECM hydrogel and screened for chemotherapy efficacy. A subset of organoids was enriched with a patient-matched immune system for screening of immunotherapy efficacy (iPTOs). Response to treatment was assessed using LIVE/DEAD staining and metabolic assays. RESULTS: Sixteen sarcomas were biofabricated into three-dimensional (3D) patient-specific sarcoma organoids with a 100% success rate. Average time from organoid development to initiation of drug testing was 7 days. Enrichment of organoids with immune system components derived from either peripheral blood mononuclear cells or lymph node cells was performed in 10/16 (62.5%) patients; 4/12 (33%) organoids did not respond to chemotherapy, while response to immunotherapy was observed in 2/10 (20%) iPTOs. CONCLUSIONS: A large subset of sarcoma organoids does not exhibit response to chemotherapy or immunotherapy, as currently seen in clinical practice. Routine development of sarcoma hydrogel-based organoids directly from the operating room is a feasible platform, allowing for such rare tumors to remain viable for personalized translational research.

Personalized Identification of Optimal HIPEC Perfusion Protocol in Patient-Derived Tumor Organoid Platform.

BACKGROUND: Chemotherapy dosing duration and perfusion temperature vary significantly in HIPEC protocols. This study investigates patient-derived tumor organoids as a platform to identify the most efficacious perfusion protocol in a personalized approach. PATIENTS AND METHODS: Peritoneal tumor tissue from 15 appendiceal and 8 colon cancer patients who underwent CRS/HIPEC were used for personalized organoid development. Organoids were perfused in parallel at 37 and 42 °C with low- and high-dose oxaliplatin (200 mg/m2 over 2 h vs. 460 mg/m2 over 30 min) and MMC (40 mg/3L over 2 h). Viability assays were performed and pooled for statistical analysis. RESULTS: An adequate organoid number was generated for 75% (6/8) of colon and 73% (11/15) of appendiceal patients. All 42 °C treatments displayed lower viability than 37 °C treatments. On pooled analysis, MMC and 200 mg/m2 oxaliplatin displayed no treatment difference for either appendiceal or colon organoids (19% vs. 25%, p = 0.22 and 27% vs. 31%, p = 0.55, respectively), whereas heated MMC was superior to 460 mg/m2 oxaliplatin in both primaries (19% vs. 54%, p < 0.001 and 27% vs. 53%, p = 0.002, respectively). In both appendiceal and colon tumor organoids, heated 200 mg/m2 oxaliplatin displayed increased cytotoxicity as compared with 460 mg/m2 oxaliplatin (25% vs. 54%, p < 0.001 and 31% vs. 53%, p = 0.008, respectively). CONCLUSIONS: Organoids treated with MMC or 200 mg/m2 heated oxaliplatin for 2 h displayed increased susceptibility in comparison with 30-min 460 mg/m2 oxaliplatin. Optimal perfusion protocol varies among patients, and organoid technology may offer a platform for tailoring HIPEC conditions to the individual patient level.

Model of Patient-Specific Immune-Enhanced Organoids for Immunotherapy Screening: Feasibility Study.

INTRODUCTION: We hypothesized that engineering a combined lymph node/melanoma organoid from the same patient would allow tumor, stroma, and immune system to remain viable for personalized immunotherapy screening. METHODS: Surgically obtained matched melanoma and lymph node biospecimens from the same patient were transferred to the laboratory and washed with saline, antibiotic, and red blood cell lysis buffer. Biospecimens were dissociated, incorporated into an extracellular matrix (ECM)-based hydrogel system, and biofabricated into three dimensional (3D) mixed melanoma/node organoids. Cells were not sorted, so as to preserve tumor heterogeneity, including stroma and immune cell components, resulting in immune-enhanced patient tumor organoids (iPTOs). Organoid sets were screened in parallel with nivolumab, pembrolizumab, ipilimumab, and dabrafenib/trametinib for 72 h. LIVE/DEAD staining and quantitative metabolism assays recorded relative drug efficacy. Histology and immunohistochemistry were used to compare tumor melanoma cells with organoid melanoma cells. Lastly, node-enhanced iPTOs were employed to activate patient-matched peripheral blood T cells for killing of tumor cells in naïve PTOs. RESULTS: Ten biospecimen sets obtained from eight stage III and IV melanoma patients were reconstructed as symbiotic immune/tumor organoids between September 2017 and June 2018. Successful establishment of viable organoid sets was 90% (9/10), although organoid yield varied with biospecimen size. Average time from organoid development to initiation of immunotherapy testing was 7 days. In three patients for whom a node was not available, it was substituted with peripheral blood mononuclear cells. iPTO response to immunotherapy was similar to specimen clinical response in 85% (6/7) patients. In an additional pilot study, peripheral T cells were circulated through iPTOs, and subsequently transferred to naïve PTOs from the same patient, resulting in tumor killing, suggesting a possible role of iPTOs in generating adaptive immunity. CONCLUSION: Development of 3D mixed immune-enhanced tumor/node organoids is a feasible platform, allowing individual patient immune system and tumor cells to remain viable for studying of personalized immunotherapy response.

Multisensor-integrated organs-on-chips platform for automated and continual in situ monitoring of organoid behaviors.

Organ-on-a-chip systems are miniaturized microfluidic 3D human tissue and organ models designed to recapitulate the important biological and physiological parameters of their in vivo counterparts. They have recently emerged as a viable platform for personalized medicine and drug screening. These in vitro models, featuring biomimetic compositions, architectures, and functions, are expected to replace the conventional planar, static cell cultures and bridge the gap between the currently used preclinical animal models and the human body. Multiple organoid models may be further connected together through the microfluidics in a similar manner in which they are arranged in vivo, providing the capability to analyze multiorgan interactions. Although a wide variety of human organ-on-a-chip models have been created, there are limited efforts on the integration of multisensor systems. However, in situ continual measuring is critical in precise assessment of the microenvironment parameters and the dynamic responses of the organs to pharmaceutical compounds over extended periods of time. In addition, automated and noninvasive capability is strongly desired for long-term monitoring. Here, we report a fully integrated modular physical, biochemical, and optical sensing platform through a fluidics-routing breadboard, which operates organ-on-a-chip units in a continual, dynamic, and automated manner. We believe that this platform technology has paved a potential avenue to promote the performance of current organ-on-a-chip models in drug screening by integrating a multitude of real-time sensors to achieve automated in situ monitoring of biophysical and biochemical parameters.

Deconstructed Microfluidic Bone Marrow On-A-Chip to Study Normal and Malignant Hemopoietic Cell-Niche Interactions.

Human hematopoietic niches are complex specialized microenvironments that maintain and regulate hematopoietic stem and progenitor cells (HSPC). Thus far, most of the studies performed investigating alterations of HSPC-niche dynamic interactions are conducted in animal models. Herein, organ microengineering with microfluidics is combined to develop a human bone marrow (BM)-on-a-chip with an integrated recirculating perfusion system that consolidates a variety of important parameters such as 3D architecture, cell-cell/cell-matrix interactions, and circulation, allowing a better mimicry of in vivo conditions. The complex BM environment is deconvoluted to 4 major distinct, but integrated, tissue-engineered 3D niche constructs housed within a single, closed, recirculating microfluidic device system, and equipped with cell tracking technology. It is shown that this technology successfully enables the identification and quantification of preferential interactions-homing and retention-of circulating normal and malignant HSPC with distinct niches.

Appendiceal Cancer Patient-Specific Tumor Organoid Model for Predicting Chemotherapy Efficacy Prior to Initiation of Treatment: A Feasibility Study.

INTRODUCTION: We have hypothesized that biofabrication of appendiceal tumor organoids allows for a more personalized clinical approach and facilitates research in a rare disease. METHODS: Appendiceal cancer specimens obtained during cytoreduction with hyperthermic intraperitoneal chemotherapy procedures (CRS/HIPEC) were dissociated and incorporated into an extracellular matrix-based hydrogel system as three-dimensional (3D), patient-specific tumor organoids. Cells were not sorted, preserving tumor heterogeneity, including stroma and immune cell components. Following establishment of organoid sets, chemotherapy drugs were screened in parallel. Live/dead staining and quantitative metabolism assays recorded which chemotherapies were most effective in killing cancer cells for a specific patient. Maintenance of cancer phenotypes were confirmed by using immunohistochemistry. RESULTS: Biospecimens from 12 patients were applied for organoid development between November 2016 and May 2018. Successful establishment rate of viable organoid sets was 75% (9/12). Average time from organoid development to chemotherapy testing was 7 days. These tumors included three high-grade appendiceal (HGA) and nine low-grade appendiceal (LGA) primaries obtained from sites of peritoneal metastasis. All tumor organoids were tested with chemotherapeutic agents exhibited responses that were either similar to the patient response or within the variability of the expected clinical response. More specifically, HGA tumor organoids derived from different patients demonstrated variable chemotherapy tumor-killing responses, whereas LGA organoids tested with the same regimens showed no response to chemotherapy. One LGA set of organoids was immune-enhanced with cells from a patient-matched lymph node to demonstrate feasibility of a symbiotic 3D reconstruction of a patient matched tumor and immune system component. CONCLUSIONS: Development of 3D appendiceal tumor organoids is feasible even in low cellularity LGA tumors, allowing for individual patient tumors to remain viable for research and personalized drug screening.

A multi-site metastasis-on-a-chip microphysiological system for assessing metastatic preference of cancer cells.

Metastatic disease remains one of the primary reasons for cancer-related deaths, yet the majority of in vitro cancer models focus on the primary tumor sites. Here, we describe a metastasis-on-a-chip device that houses multiple bioengineered three-dimensional (3D) organoids, established by a 3D photopatterning technique employing extracellular matrix-derived hydrogel biomaterials. Specifically, cancer cells begin in colorectal cancer (CRC) organoid, which resides in a single microfluidic chamber connected to multiple downstream chambers in which liver, lung, and endothelial constructs are housed. Under recirculating fluid flow, tumor cells grow in the primary site, eventually enter circulation, and can be tracked via fluorescent imaging. Importantly, we describe that in the current version of this platform, HCT116 CRC cells preferentially home to the liver and lung constructs; the corresponding organs of which CRC metastases arise the most in human patients. We believe that in subsequent studies this platform can be implemented to better understand the mechanisms underlying metastasis, perhaps resulting in the identification of targets for intervention.

A reductionist metastasis-on-a-chip platform for in vitro tumor progression modeling and drug screening.

Current animal and 2-D cell culture models employed in metastasis research and drug discovery remain poor mimics of human cancer physiology. Here we describe a "metastasis-on-a-chip" system allowing real time tracking of fluorescent colon cancer cells migrating from hydrogel-fabricated gut constructs to downstream liver constructs within a circulatory fluidic device system that responds to environmental manipulation and drug treatment. Devices consist of two chambers in which gut and liver constructs are housed independently, but are connected in series via circulating fluid flow. Constructs were biofabricated with a hyaluronic acid-based hydrogel system, capable of a variety of customizations, inside of which representative host tissue cells were suspended and metastatic colon carcinoma tumor foci were created. The host tissue of the constructs expressed normal epithelial markers, which the tumor foci failed to express. Instead, tumor regions lost membrane-bound adhesion markers, and expressed mesenchymal and proliferative markers, suggesting a metastatic phenotype. Metastatic tumor foci grew in size, eventually disseminating from the intestine construct and entering circulation, subsequently reaching in the liver construct, thus mimicking some of the migratory events observed during metastasis. Lastly, we demonstrated the ability to manipulate the system, including chemically modulating the hydrogel system mechanical properties and administering chemotherapeutic agents, and evaluated the effects of these parameters on invasive tumor migration. These results describe the capability of this early stage metastasis-on-a-chip system to model several important characteristics of human metastasis, thereby demonstrating the potential of the platform for making meaningful advances in cancer investigation and drug discovery. Biotechnol. Bioeng. 2016;113: 2020-2032. © 2016 Wiley Periodicals, Inc.

Bioengineered in vitro three-dimensional tumor models in endocrine cancers.

Abstract: Endocrine tumors are a heterogeneous cluster of malignancies that originate from cells that can secrete hormones. Examples include, but are not limited to, thyroid cancer, adrenocortical carcinoma, and neuroendocrine tumors. Many endocrine tumors are relatively slow to proliferate, and as such, they often do not respond well to common antiproliferative chemotherapies. Therefore, increasing attention has been given to targeted therapies and immunotherapies in these diseases. However, in contrast to other cancers, many endocrine tumors are relatively rare, and as a result, less is understood about their biology, including specific targets for intervention. Our limited understanding of such tumors is in part due to a limitation in model systems that accurately recapitulate and enable mechanistic exploration of these tumors. While mouse models and 2D cell cultures exist for some endocrine tumors, these models often may not accurately model nuances of human endocrine tumors. Mice differ from human endocrine physiology and 2D cell cultures fail to recapitulate the heterogeneity and 3D architectures of in vivo tumors. To complement these traditional cancer models, bioengineered 3D tumor models, such as organoids and tumor-on-a-chip systems, have advanced rapidly in the past decade. However, these technologies have only recently been applied to most endocrine tumors. In this review we provide descriptions of these platforms, focusing on thyroid, adrenal, and neuroendocrine tumors and how they have been and are being applied in the context of endocrine tumors.

An osteosarcoma-on-a-chip model for studying osteosarcoma matrix-cell interactions and drug responses.

Marrow niches in osteosarcoma (OS) are a specialized microenvironment that is essential for the maintenance and regulation of OS cells. However, existing animal xenograft models are plagued by variability, complexity, and high cost. Herein, we used a decellularized osteosarcoma extracellular matrix (dOsEM) loaded with extracellular vesicles from human bone marrow-derived stem cells (hBMSC-EVs) and OS cells as a bioink to construct a micro-osteosarcoma (micro-OS) through 3D printing. The micro-OS was further combined with a microfluidic system to develop into an OS-on-a-chip (OOC) with a built-in recirculating perfusion system. The OOC system successfully integrated bone marrow niches, cell‒cell and cell-matrix crosstalk, and circulation, allowing a more accurate representation of OS characteristics in vivo. Moreover, the OOC system may serve as a valuable research platform for studying OS biological mechanisms compared with traditional xenograft models and is expected to enable precise and rapid evaluation and consequently more effective and comprehensive treatments for OS.

Cancer Cell Targeting, Magnetic Sorting, and SERS Detection through Cell Surface Receptors.

Integrins are cellular surface receptors responsible for the activation of many cellular pathways in cancer. These integrin proteins can be specifically targeted by small peptide sequences that offer the potential for the differentiation of cellular subpopulations by using magnetically assisted cellular sorting techniques. By adding a gold shell to the magnetic nanoparticles, these integrin-peptide interactions can be differentiated by surface-enhanced Raman spectroscopy (SERS), providing a quick and reliable method for on-target binding. In this paper, we demonstrate the ability to differentiate the peptide-protein interactions of the small peptides CDPGYIGSR and cyclic RGDfC functionalized on gold-coated magnetic nanoparticles with the integrins they are known to bind to using their SERS signal. SW480 and SW620 colorectal cancer cells known to have the integrins of interest were then magnetically sorted using these functionalized nanoparticles, suggesting differentiation between the sorted populations and integrin populations among the two cell lines.

A 3D adrenocortical carcinoma tumor platform for preclinical modeling of drug response and matrix metalloproteinase activity.

Adrenocortical carcinoma (ACC) has a poor prognosis, and no new drugs have been identified in decades. The absence of drug development can partly be attributed to a lack of preclinical models. Both animal models and 2D cell cultures of ACC fail to accurately mimic the disease, as animal physiology is inherently different than humans, and 2D cultures fail to represent the crucial 3D architecture. Organoids and other small 3D in vitro models of tissues or tumors can model certain complexities of human in vivo biology; however, this technology has largely yet to be applied to ACC. In this study, we describe the generation of 3D tumor constructs from an established ACC cell line, NCI-H295R. NCI-H295R cells were encapsulated to generate 3D ACC constructs. Tumor constructs were assessed for biomarker expression, viability, proliferation, and cortisol production. In addition, matrix metalloproteinase (MMP) functionality was assessed directly using fluorogenic MMP-sensitive biosensors and through infusion of NCI-H295R cells into a metastasis-on-a-chip microfluidic device platform. ACC tumor constructs showed expression of biomarkers associated with ACC, including SF-1, Melan A, and inhibin alpha. Treatment of ACC tumor constructs with chemotherapeutics demonstrated decreased drug sensitivity compared to 2D cell culture. Since most tumor cells migrate through tissue using MMPs to break down extracellular matrix, we validated the utility of ACC tumor constructs by integrating fluorogenic MMP-sensitive peptide biosensors within the tumor constructs. Lastly, in our metastasis-on-a-chip device, NCI-H295R cells successfully engrafted in a downstream lung cell line-based construct, but invasion distance into the lung construct was decreased by MMP inhibition. These studies, which would not be possible using 2D cell cultures, demonstrated that NCI-H295R cells secreted active MMPs that are used for invasion in 3D. This work represents the first evidence of a 3D tumor constructs platform for ACC that can be deployed for future mechanistic studies as well as development of new targets for intervention and therapies.

A 3D adrenocortical carcinoma tumor platform for preclinical modeling of drug response and matrix metalloproteinase activity.

Adrenocortical carcinoma (ACC) has a poor prognosis, and no new drugs have been identified in decades. The absence of drug development can partly be attributed to a lack of preclinical models. Both animal models and 2D cell cultures of ACC fail to accurately mimic the disease, as animal physiology is inherently different than humans, and 2D cultures fail to represent the crucial 3D architecture. Organoids and other small 3D in vitro models of tissues or tumors can model certain complexities of human in vivo biology; however, this technology has largely yet to be applied to ACC. In this study, we describe the generation of 3D tumor constructs from an established ACC cell line, NCI-H295R. NCI-H295R cells were encapsulated to generate 3D ACC constructs. Tumor constructs were assessed for biomarker expression, viability, proliferation, and cortisol production. In addition, matrix metalloproteinase (MMP) functionality was assessed directly using fluorogenic MMP-sensitive biosensors and through infusion of NCI-H295R cells into a metastasis-on-a-chip microfluidic device platform. ACC tumor constructs showed expression of biomarkers associated with ACC, including SF-1, Melan A, and inhibin α. Treatment of ACC tumor constructs with chemotherapeutics demonstrated decreased drug sensitivity compared to 2D cell culture. Since most tumor cells migrate through tissue using MMPs to break down extracellular matrix, we validated the utility of ACC tumor constructs by integrating fluorogenic MMP-sensitive peptide biosensors within the tumor constructs. Lastly, in our metastasis-on-a-chip device, NCI-H295R cells successfully engrafted in a downstream lung cell line-based construct, but invasion distance into the lung construct was decreased by MMP inhibition. These studies, which would not be possible using 2D cell cultures, demonstrated that NCI-H295R cells secreted active MMPs that are used for invasion in 3D. This work represents the first evidence of a 3D tumor constructs platform for ACC that can be deployed for future mechanistic studies as well as development of new targets for intervention and therapies.

Detection of lineage-reprogramming efficiency of tumor cells in a 3D-printed liver-on-a-chip model.

Background: The liver metastasis accompanied with the loss of liver function is one of the most common complications in patients with triple-negative breast cancers (TNBC). Lineage reprogramming, as a technique direct inducing the functional cell types from one lineage to another lineage without passing through an intermediate pluripotent stage, is promising in changing cell fates and overcoming the limitations of primary cells. However, most reprogramming techniques are derived from human fibroblasts, and whether cancer cells can be reversed into hepatocytes remains elusive. Methods: Herein, we simplify preparation of reprogramming reagents by expressing six transcriptional factors (HNF4A, FOXA2, FOXA3, ATF5, PROX1, and HNF1) from two lentiviral vectors, each expressing three factors. Then the virus was transduced into MDA-MB-231 cells to generated human induced hepatocyte-like cells (hiHeps) and single-cell sequencing was used to analyze the fate for the cells after reprogramming. Furthermore, we constructed a Liver-on-a-chip (LOC) model by bioprinting the Gelatin Methacryloyl hydrogel loaded with hepatocyte extracellular vesicles (GelMA-EV) bioink onto the microfluidic chip to assess the metastasis behavior of the reprogrammed TNBC cells under the 3D liver microenvironment in vitro. Results: The combination of the genes HNF4A, FOXA2, FOXA3, ATF5, PROX1 and HNF1A could reprogram MDA-MB-231 tumor cells into human-induced hepatocytes (hiHeps), limiting metastasis of these cells. Single-cell sequencing analysis showed that the oncogenes were significantly inhibited while the liver-specific genes were activated after lineage reprogramming. Finally, the constructed LOC model showed that the hepatic phenotypes of the reprogrammed cells could be observed, and the metastasis of embedded cancer cells could be inhibited under the liver microenvironment. Conclusion: Our findings demonstrate that reprogramming could be a promising method to produce hepatocytes and treat TNBC liver metastasis. And the LOC model could intimate the 3D liver microenvironment and assess the behavior of the reprogrammed TNBC cells.

Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with high metastasis and therapeutic resistance. Activating transcription factor 4 (ATF4), a master regulator of cellular stress, is exploited by cancer cells to survive. Prior research and data reported provide evidence that high ATF4 expression correlates with worse overall survival in PDAC. Tomatidine, a natural steroidal alkaloid, is associated with inhibition of ATF4 signaling in multiple diseases. Here, we discovered that in vitro and in vivo tomatidine treatment of PDAC cells inhibits tumor growth. Tomatidine inhibited nuclear translocation of ATF4 and reduced the transcriptional binding of ATF4 with downstream promoters. Tomatidine enhanced gemcitabine chemosensitivity in 3D ECM-hydrogels and in vivo. Tomatidine treatment was associated with induction of ferroptosis signaling validated by increased lipid peroxidation, mitochondrial biogenesis, and decreased GPX4 expression in PDAC cells. This study highlights a possible therapeutic approach utilizing a plant-derived metabolite, tomatidine, to target ATF4 activity in PDAC.

Strategies for developing complex multi-component in vitro tumor models: Highlights in glioblastoma.

In recent years, many research groups have begun to utilize bioengineered in vitro models of cancer to study mechanisms of disease progression, test drug candidates, and develop platforms to advance personalized drug treatment options. Due to advances in cell and tissue engineering over the last few decades, there are now a myriad of tools that can be used to create such in vitro systems. In this review, we describe the considerations one must take when developing model systems that accurately mimic the in vivo tumor microenvironment (TME) and can be used to answer specific scientific questions. We will summarize the importance of cell sourcing in models with one or multiple cell types and outline the importance of choosing biomaterials that accurately mimic the native extracellular matrix (ECM) of the tumor or tissue that is being modeled. We then provide examples of how these two components can be used in concert in a variety of model form factors and conclude by discussing how biofabrication techniques such as bioprinting and organ-on-a-chip fabrication can be used to create highly reproducible complex in vitro models. Since this topic has a broad range of applications, we use the final section of the review to dive deeper into one type of cancer, glioblastoma, to illustrate how these components come together to further our knowledge of cancer biology and move us closer to developing novel drugs and systems that improve patient outcomes.