Microphysiological systems for solid tumor immunotherapy: opportunities and challenges.

Immunotherapy remains more effective for hematologic tumors than for solid tumors. One of the main challenges to immunotherapy of solid tumors is the immunosuppressive microenvironment these tumors generate, which limits the cytotoxic capabilities of immune effector cells (e.g., cytotoxic T and natural killer cells). This microenvironment is characterized by hypoxia, nutrient starvation, accumulated waste products, and acidic pH. Tumor-hijacked cells, such as fibroblasts, macrophages, and T regulatory cells, also contribute to this inhospitable microenvironment for immune cells by secreting immunosuppressive cytokines that suppress the antitumor immune response and lead to immune evasion. Thus, there is a strong interest in developing new drugs and cell formulations that modulate the tumor microenvironment and reduce tumor cell immune evasion. Microphysiological systems (MPSs) are versatile tools that may accelerate the development and evaluation of these therapies, although specific examples showcasing the potential of MPSs remain rare. Advances in microtechnologies have led to the development of sophisticated microfluidic devices used to recapitulate tumor complexity. The resulting models, also known as microphysiological systems (MPSs), are versatile tools with which to decipher the molecular mechanisms driving immune cell antitumor cytotoxicity, immune cell exhaustion, and immune cell exclusion and to evaluate new targeted immunotherapies. Here, we review existing microphysiological platforms to study immuno-oncological applications and discuss challenges and opportunities in the field.

Microphysiological model reveals the promise of memory-like natural killer cell immunotherapy for HIV± cancer.

Numerous studies are exploring the use of cell adoptive therapies to treat hematological malignancies as well as solid tumors. However, there are numerous factors that dampen the immune response, including viruses like human immunodeficiency virus. In this study, we leverage human-derived microphysiological models to reverse-engineer the HIV-immune system interaction and evaluate the potential of memory-like natural killer cells for HIV+ head and neck cancer, one of the most common tumors in patients living with human immunodeficiency virus. Here, we evaluate multiple aspects of the memory-like natural killer cell response in human-derived bioengineered environments, including immune cell extravasation, tumor penetration, tumor killing, T cell dependence, virus suppression, and compatibility with retroviral medication. Overall, these results suggest that memory-like natural killer cells are capable of operating without T cell assistance and could simultaneously destroy head and neck cancer cells as well as reduce viral latency.

Griddient: a microfluidic array to generate reconfigurable gradients on-demand for spatial biology applications.

Biological tissues are highly organized structures where spatial-temporal gradients (e.g., nutrients, hypoxia, cytokines) modulate multiple physiological and pathological processes including inflammation, tissue regeneration, embryogenesis, and cancer progression. Current in vitro technologies struggle to capture the complexity of these transient microenvironmental gradients, do not provide dynamic control over the gradient profile, are complex and poorly suited for high throughput applications. Therefore, we have designed Griddent, a user-friendly platform with the capability of generating controllable and reversible gradients in a 3D microenvironment. Our platform consists of an array of 32 microfluidic chambers connected to a 384 well-array through a diffusion port at the bottom of each reservoir well. The diffusion ports are optimized to ensure gradient stability and facilitate manual micropipette loading. This platform is compatible with molecular and functional spatial biology as well as optical and fluorescence microscopy. In this work, we have used this platform to study cancer progression.

A role for microfluidic systems in precision medicine.

Precision oncology continues to challenge the "one-size-fits-all" dogma. Under the precision oncology banner, cancer patients are screened for molecular tumor alterations that predict treatment response, ideally leading to optimal treatments. Functional assays that directly evaluate treatment efficacy on the patient's cells offer an alternative and complementary tool to improve the accuracy of precision oncology. Unfortunately, traditional Petri dish-based assays overlook much tumor complexity, limiting their potential as predictive functional biomarkers. Here, we review past applications of microfluidic systems for precision medicine and discuss the present and potential future role of functional microfluidic assays as treatment predictors.

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

Models of Renal Cell Carcinoma Used to Investigate Molecular Mechanisms and Develop New Therapeutics.

Modeling renal cell carcinoma is critical to investigating tumor biology and therapeutic mechanisms. Multiple systems have been developed to represent critical components of the tumor and its surrounding microenvironment. Prominent in vitro models include traditional cell cultures, 3D organoid models, and microphysiological devices. In vivo models consist of murine patient derived xenografts or genetically engineered mice. Each system has unique advantages as well as limitations and researchers must thoroughly understand each model to properly investigate research questions. This review addresses common model systems for renal cell carcinoma and critically evaluates their performance and ability to measure tumor characteristics.

Microphysiological model of renal cell carcinoma to inform anti-angiogenic therapy.

Renal cell carcinomas are common genitourinary tumors characterized by high vascularization and strong reliance on glycolysis. Despite the many available therapies for renal cell carcinomas, first-line targeted therapies, such as cabozantinib, and durable reaponses are seen in only a small percentage of patients. Yet, little is known about the mechanisms that drive response (or lack thereof). This dearth of knowledge can be explained by the dynamic and complex microenvironment of renal carcinoma, which remains challenging to recapitulate in vitro. Here, we present a microphysiological model of renal cell carcinoma, including a tubular blood vessel model of induced pluripotent stem cell-derived endothelial cells and an adjacent 3D carcinoma model. Our model recapitulated hypoxia, glycolic metabolism, and sprouting angiogenesis. Using our model, we showed that cabozantinib altered cancer cell metabolism and decreased sprouting angiogenesis but did not restore barrier function. This microphysiological model could be helpful to elucidate, through multiple endpoints, the contributions of the relevant environmental components in eliciting a functional response or resistance to therapy in renal cell carcinoma.

Primary head and neck tumour-derived fibroblasts promote lymphangiogenesis in a lymphatic organotypic co-culture model.

BACKGROUND: In head and neck cancer, intratumour lymphatic density and tumour lymphangiogenesis have been correlated with lymphatic metastasis, making lymphangiogenesis a promising therapeutic target. However, inter-patient tumour heterogeneity makes it challenging to predict tumour progression and lymph node metastasis. Understanding the lymphangiogenic-promoting factors leading to metastasis (e.g., tumour-derived fibroblasts or TDF), would help develop strategies to improve patient outcomes. METHODS: A microfluidic in vitro model of a tubular lymphatic vessel was co-cultured with primary TDF from head and neck cancer patients to evaluate the effect of TDF on lymphangiogenesis. We assessed the length and number of lymphangiogenic sprouts and vessel permeability via microscopy and image analysis. Finally, we characterised lymphatic vessel conditioning by TDF via RT-qPCR. FINDINGS: Lymphatic vessels were conditioned by the TDF in a patient-specific manner. Specifically, the presence of TDF induced sprouting, altered vessel permeability, and increased the expression of pro-lymphangiogenic genes. Gene expression and functional responses in the fibroblast-conditioned lymphatic vessels were consistent with the patient tumour stage and lymph node status. IGF-1, upregulated among patients, was targeted to validate our personalised medicine approach. Interestingly, IGF-1 blockade was not effective across different patients. INTERPRETATION: The use of lymphatic organotypic models incorporating head and neck TDF provides insight into the pathways leading to lymphangiogenesis in each patient. This model provided a platform to test anti-angiogenic therapeutics and inform of their effectiveness for individual patients. FUNDING: NIH R33CA225281. Wisconsin Head and Neck SPORE NIH P50DE026787. NIH R01AI34749.

Toward improved in vitro models of human cancer.

Cancer is a leading cause of death across the world and continues to increase in incidence. Despite years of research, multiple tumors (e.g., glioblastoma, pancreatic cancer) still have limited treatment options in the clinic. Additionally, the attrition rate and cost of drug development have continued to increase. This trend is partly explained by the poor predictive power of traditional in vitro tools and animal models. Moreover, multiple studies have highlighted that cell culture in traditional Petri dishes commonly fail to predict drug sensitivity. Conversely, animal models present differences in tumor biology compared with human pathologies, explaining why promising therapies tested in animal models often fail when tested in humans. The surging complexity of patient management with the advent of cancer vaccines, immunotherapy, and precision medicine demands more robust and patient-specific tools to better inform our understanding and treatment of human cancer. Advances in stem cell biology, microfluidics, and cell culture have led to the development of sophisticated bioengineered microscale organotypic models (BMOMs) that could fill this gap. In this Perspective, we discuss the advantages and limitations of patient-specific BMOMs to improve our understanding of cancer and how these tools can help to confer insight into predicting patient response to therapy.

Microfluidic tumor-on-a-chip model to evaluate the role of tumor environmental stress on NK cell exhaustion.

Solid tumors generate a suppressive environment that imposes an overwhelming burden on the immune system. Nutrient depletion, waste product accumulation, hypoxia, and pH acidification severely compromise the capacity of effector immune cells such as T and natural killer (NK) cells to destroy cancer cells. However, the specific molecular mechanisms driving immune suppression, as well as the capacity of immune cells to adapt to the suppressive environment, are not completely understood. Thus, here, we used an in vitro microfluidic tumor-on-a-chip platform to evaluate how NK cells respond to the tumor-induced suppressive environment. The results demonstrated that the suppressive environment created by the tumor gradually eroded NK cell cytotoxic capacity, leading to compromised NK cell surveillance and tumor tolerance. Further, NK cell exhaustion persisted for an extended period of time after removing NK cells from the microfluidic platform. Last, the addition of checkpoint inhibitors and immunomodulatory agents alleviated NK cell exhaustion.

Elucidating cancer-vascular paracrine signaling using a human organotypic breast cancer cell extravasation model.

In cancer metastasis, extravasation refers to the process where tumor cells exit the bloodstream by crossing the endothelium and invade the surrounding tissue. Tumor cells engage in complex crosstalk with other active players such as the endothelium leading to changes in functional behavior that exert pro-extravasation effects. Most in vitro studies to date have only focused on the independent effects of molecular targets on the functional changes of cancer cell extravasation behavior. However, singular targets cannot combat complex interactions involved in tumor cell extravasation that affects multiple cell types and signaling pathways. In this study, we employ an organotypic microfluidic model of human vasculature to investigate the independent and combined role of multiple upregulated secreted factors resulting from cancer-vascular interactions during cancer cell extravasation. The device consists of a tubular endothelial vessel generated from induced pluripotent stem cell derived endothelial cells within a collagen-fibrinogen matrix with breast cancer cells injected through and cultured along the lumen of the vessel. Our system identified cancer-vascular crosstalk, involving invasive breast cancer cells, that results in increased levels of secreted IL-6, IL-8, and MMP-3. Our model also showed that upregulation of these secreted factors correlates with invasive/metastatic potential of breast cancer cells. We also used therapeutic inhibitors to assess the independent and combined role of multiple signaling factors on the overall changes in functional behavior of both the cancer cells and the endothelium that promote extravasation. Taken together, these results demonstrate the potential of our organotypic model in elucidating mechanisms through which cancer-vascular interactions can promote extravasation, and in conducting functional assessment of therapeutic drugs that prevent extravasation in cancer metastasis.

Microfluidic Tumor-on-a-Chip Model to Study Tumor Metabolic Vulnerability.

Tumor-specific metabolic adaptations offer an interesting therapeutic opportunity to selectively destroy cancer cells. However, solid tumors also present gradients of nutrients and waste products across the tumor mass, forcing tumor cells to adapt their metabolism depending on nutrient availability in the surrounding microenvironment. Thus, solid tumors display a heterogenous metabolic phenotype across the tumor mass, which complicates the design of effective therapies that target all the tumor populations present. In this work, we used a microfluidic device to study tumor metabolic vulnerability to several metabolic inhibitors. The microdevice included a central chamber to culture tumor cells in a three-dimensional (3D) matrix, and a lumen in one of the chamber flanks. This design created an asymmetric nutrient distribution across the central chamber, generating gradients of cell viability. The results revealed that tumor cells located in a nutrient-enriched environment showed low to no sensitivity to metabolic inhibitors targeting glycolysis, fatty acid oxidation, or oxidative phosphorylation. Conversely, when cell density inside of the model was increased, compromising nutrient supply, the addition of these metabolic inhibitors disrupted cellular redox balance and led to tumor cell death.

Organotypic primary blood vessel models of clear cell renal cell carcinoma for single-patient clinical trials.

Clear cell renal cell carcinoma (ccRCC) is a common genitourinary cancer associated with the development of abnormal tumor angiogenesis. Although multiple anti-angiogenic therapies have been developed, responses to individual treatment are highly variable between patients. Thus, the use of one-patient clinical trials has been suggested as an alternative to standard trials. We used a microfluidic device to generate organotypic primary patient-specific blood vessel models using normal (NEnC) and tumor-associated primary CD31+ selected cells (TEnC). Our model was able to recapitulate differences in angiogenic sprouting and vessel permeability that characterize normal and tumor-associated vessels. We analyzed the expression profile of vessel models to define vascular normalization in a patient-specific manner. Using this data, we identified actionable targets to normalize TEnC vessel function to a more NEnC-like phenotype. Finally, we tested two of these drugs in our patient-specific models to determine the efficiency in restoring vessel function showing the potential of the model for single-patient clinical trials.

Breast Fibroblasts and ECM Components Modulate Breast Cancer Cell Migration Through the Secretion of MMPs in a 3D Microfluidic Co-Culture Model.

The extracellular matrix (ECM) composition greatly influences cancer progression, leading to differential invasion, migration, and metastatic potential. In breast cancer, ECM components, such as fibroblasts and ECM proteins, have the potential to alter cancer cell migration. However, the lack of in vitro migration models that can vary ECM composition limits our knowledge of how specific ECM components contribute to cancer progression. Here, a microfluidic model was used to study the effect of 3D heterogeneous ECMs (i.e., fibroblasts and different ECM protein compositions) on the migration distance of a highly invasive human breast cancer cell line, MDA-MB-231. Specifically, we show that in the presence of normal breast fibroblasts, a fibronectin-rich matrix induces more cancer cell migration. Analysis of the ECM revealed the presence of ECM tunnels. Likewise, cancer-stromal crosstalk induced an increase in the secretion of metalloproteinases (MMPs) in co-cultures. When MMPs were inhibited, migration distance decreased in all conditions except for the fibronectin-rich matrix in the co-culture with human mammary fibroblasts (HMFs). This model mimics the in vivo invasion microenvironment, allowing the examination of cancer cell migration in a relevant context. In general, this data demonstrates the capability of the model to pinpoint the contribution of different components of the tumor microenvironment (TME).

Matrix density drives 3D organotypic lymphatic vessel activation in a microfluidic model of the breast tumor microenvironment.

Lymphatic vessels (LVs) have been suggested as a preferential conduit for metastatic progression in breast cancer, where a correlation between the occurrence of lymph node metastasis and an increased extracellular matrix (ECM) density has been reported. However, the effect of ECM density on LV function is largely unknown. To better understand these effects, we used a microfluidic device to recreate tubular LVs in a collagen type I matrix. The density of the matrix was tailored to mimic normal breast tissue using a low-density collagen (LD-3 mg mL-1) and cancerous breast tissue using a high-density collagen (HD-6 mg mL-1). We investigated the effect of ECM density on LV morphology, growth, cytokine secretion, and barrier function. LVs cultured in HD matrices showed morphological changes as compared to LVs cultured in a LD matrix. Specifically, LVs cultured in HD matrices had a 3-fold higher secretion of the pro-inflammatory cytokine, IL-6, and a leakier phenotype, suggesting LVs acquired characteristics of activated vessels. Interestingly, LV leakiness was mitigated by blocking the IL-6 receptor on the lymphatic ECs, maintaining endothelium permeability at similar levels of LV cultured in a LD matrix. To recreate a more in vivo microenvironment, we incorporated metastatic breast cancer cells (MDA-MB-231) into the LD and HD matrices. For HD matrices, co-culture with MDA-MB-231 cells exacerbated vessel leakiness and secretion of IL-6. In summary, our data suggest that (1) ECM density is an important microenvironmental cue that affects LV function in the breast tumor microenvironment (TME), (2) dense matrices condition LVs towards an activated phenotype and (3) blockade of IL-6 signaling may be a potential therapeutic target to mitigate LV dysfunction. Overall, modeling LVs and their interactions with the TME can help identify novel therapeutic targets and, in turn, advance therapeutic discovery.

Author Correction: Enabling cell recovery from 3D cell culture microfluidic devices for tumour microenvironment biomarker profiling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Development of a Microfluidic Array to Study Drug Response in Breast Cancer.

Luminal geometries are common structures in biology, which are challenging to mimic using conventional in vitro techniques based on the use of Petri dishes. In this context, microfluidic systems can mimic the lumen geometry, enabling a large variety of studies. However, most microfluidic models still rely on polydimethylsiloxane (PDMS), a material that is not amenable for high-throughput fabrication and presents some limitations compared with other materials such as polystyrene. Thus, we have developed a microfluidic device array to generate multiple bio-relevant luminal structures utilizing polystyrene and micro-milling. This platform offers a scalable alternative to conventional microfluidic devices designed in PDMS. Additionally, the use of polystyrene has well described advantages, such as lower permeability to hydrophobic molecules compared with PDMS, while maintaining excellent viability and optical properties. Breast cancer cells cultured in the devices exhibited high cell viability similar to PDMS-based microdevices. Further, co-culture experiments with different breast cell types showed the potential of the model to study breast cancer invasion. Finally, we demonstrated the potential of the microfluidic array for drug screening, testing chemotherapy drugs and photodynamic therapy agents for breast cancer.

Tumor-on-a-chip: a microfluidic model to study cell response to environmental gradients.

Limited blood supply and rapid tumor metabolism within solid tumors leads to nutrient starvation, waste product accumulation and the generation of pH gradients across the tumor mass. These environmental conditions modify multiple cellular functions, including metabolism, proliferation, and drug response. However, capturing the spatial metabolic and phenotypic heterogeneity of the tumor with classic in vitro models remains challenging. Thus, in this work a microfluidic tumor slice model was developed to study cell behavior under metabolic starvation gradients. The presented microdevice comprises a central chamber where tumor cells were cultured in a 3D collagen hydrogel. A lumen on the flank of the chamber was used to perfuse media, mimicking the vasculature. Under these circumstances, tumor cell metabolism led to the generation of viability, proliferation and pH gradients. The model decoupled the influence of oxygen from other nutrients, revealing that cell necrosis at the core of the model could be explained by nutrient starvation. The microdevice can be disassembled to retrieve the cells from the desired locations to study molecular adaptions due to nutrient starvation. When exposed to these pH gradients and low nutrient conditions, cancer cells showed multiple changes in their gene expression profile depending on their distance from the lumen. Those cells located further from the lumen upregulated several genes related to stress and survival response, whereas genes related to proliferation and DNA repair were downregulated. This model may help to identify new therapeutic opportunities to target the metabolic heterogeneity observed in solid tumors.

Modulation of Antioxidant Potential with Coenzyme Q10 Suppressed Invasion of Temozolomide-Resistant Rat Glioma In Vitro and In Vivo.

The main reasons for the inefficiency of standard glioblastoma (GBM) therapy are the occurrence of chemoresistance and the invasion of GBM cells into surrounding brain tissues. New therapeutic approaches obstructing these processes may provide substantial survival improvements. The purpose of this study was to assess the potential of lipophilic antioxidant coenzyme Q10 (CoQ10) as a scavenger of reactive oxygen species (ROS) to increase sensitivity to temozolomide (TMZ) and suppress glioma cell invasion. To that end, we used a previously established TMZ-resistant RC6 rat glioma cell line, characterized by increased production of ROS, altered antioxidative capacity, and high invasion potential. CoQ10 in combination with TMZ exerted a synergistic antiproliferative effect. These results were confirmed in a 3D model of microfluidic devices showing that the CoQ10 and TMZ combination is more cytotoxic to RC6 cells than TMZ monotherapy. In addition, cotreatment with TMZ increased expression of mitochondrial antioxidant enzymes in RC6 cells. The anti-invasive potential of the combined treatment was shown by gelatin degradation, Matrigel invasion, and 3D spheroid invasion assays as well as in animal models. Inhibition of MMP9 gene expression as well as decreased N-cadherin and vimentin protein expression implied that CoQ10 can suppress invasiveness and the epithelial to mesenchymal transition in RC6 cells. Therefore, our data provide evidences in favor of CoQ10 supplementation to standard GBM treatment due to its potential to inhibit GBM invasion through modulation of the antioxidant capacity.