Tackling the tumor microenvironment - how can complex tumor models in vitro aid oncology drug development?

INTRODUCTION: Identifying effective cancer drugs remains an inefficient process. Drug efficacy in traditional preclinical cancer models translates poorly into therapy in the clinic. Implementation of preclinical models that incorporate the tumor microenvironment (TME) is needed to improve selection of active drugs prior to clinical trials. AREAS COVERED: Progression of cancer results from the behavior of cancer cells in concert with the host's histopathological background. Nonetheless, complex preclinical models with a relevant microenvironment have yet to become an integral part of drug development. This review discusses existing models and provides a synopsis of active areas of cancer drug development where implementation would be of value. Their contribution to finding therapeutics in immune oncology, angiogenesis, regulated cell death and targeting tumor fibroblasts as well as optimization of drug delivery, combination therapy, and biomarkers of efficacy is considered. EXPERT OPINION: Complex tumor models in vitro (CTMIVs) that mimic the organotypic architecture of neoplastic tumors have boosted research into TME influence on traditional cytoreductive chemotherapy as well as the detection of specific TME targets. Despite advances in technical prowess, CTMIVs can only address specific aspects of cancer pathophysiology.

Pathophysiologic and Pharmacologic Considerations to Improve the Design and Application of Antibody-Drug Conjugates.

Antibody-drug conjugates (ADC) have emerged as one of the pillars of clinical disease management in oncology. The biggest hurdle to widespread development and application of ADCs has been a narrow therapeutic index. Advances in antibody technologies and formats as well as novel linker and payload chemistries have begun to facilitate structural improvements to ADCs. However, the interplay of structural characteristics with physiologic and pharmacologic factors determining therapeutic success has garnered less attention. This review elaborates on the pharmacology of ADCs, the pathophysiology of cancerous tissues, and the reciprocal consequences on ADC properties and functions. While most currently approved ADCs utilize either microtubule inhibition or DNA damage as primary mechanisms of action, we present arguments to expand this repertoire and highlight the need for payload mechanisms that exploit disease-specific vulnerabilities. We promote the idea that the choice of antibody format, targeting antigen, linker properties, and payload of an ADC should be deliberately fit for purpose by taking the pathophysiology of disease and the specific pharmacology of the drug entity into account, thus allowing a higher probability of clinical success.

Application of LDH assay for therapeutic efficacy evaluation of ex vivo tumor models.

The current standard preclinical oncology models are not able to fully recapitulate therapeutic targets and clinically relevant disease biology, evidenced by the 90% attrition rate of new therapies in clinical trials. Three-dimensional (3D) culture systems have the potential to enhance the relevance of preclinical models. However, the limitations of currently available cellular assays to accurately evaluate therapeutic efficacy in these models are hindering their widespread adoption. We assessed the compatibility of the lactate dehydrogenase (LDH) assay in 3D spheroid cultures against other commercially available readout methods. We developed a standardized protocol to apply the LDH assay to ex vivo cultures, considering the impact of culture growth dynamics. We show that accounting for growth rates and background release levels of LDH are sufficient to make the LDH assay a suitable methodology for longitudinal monitoring and endpoint assessment of therapeutic efficacy in both cell line-derived xenografts (xenospheres) and patient-derived explant cultures. This method has the added value of being non-destructive and not dependent on reagent penetration or manipulation of the parent material. The establishment of reliable readout methods for complex 3D culture systems will further the utility of these tumor models in preclinical and co-clinical drug development studies.

Effects of culture condition on epigenomic profiles of brain tumor cells.

Personalized cancer medicine offers the promise of more effective treatments that are tailored to an individual's own dynamic cancer phenotype. Meanwhile, tissue-engineering approaches to modeling tumors may complement these advances by providing a powerful new approach to understanding the adaptation dynamics occurring during treatment. However, in both of these areas new tools will be required to gain a full picture of the genetic and epigenetic regulators of phenotype dynamics occurring in the small populations of cells that drive resistance. In this study, we perform epigenomic analysis of brain tumor cells that are collected from micro-engineered three-dimensional tumor models, overcoming the challenges associated with the small numbers of cells contained within these micro-tissue niches, in this case collecting ~1,000 cells per sample. Specifically, we use a high-resolution epigenomic analysis method known as microfluidic-oscillatory-washing-based chromatin immunoprecipitation with sequencing (MOWChIP-seq) to analyze histone methylation patterns (H3K4me3). We identified gene loci that are associated with the H3K4me3 modification, which is generally a mark of active transcription. We compared methylation patterns in standard 2D cultures and 3D cultures based on type I collagen hydrogels, under both normoxic and hypoxic conditions. We found that culture dimensionality drastically impacted the H3k4me3 profile and resulted in differential modifications in response to hypoxic stress. Differentially H3K4me3-marked regions under the culture conditions used in this study have important implications for gene expression differences that have been previously observed. In total, our work illustrates a direct connection between cell culture or tissue niche condition and genome-wide alterations in histone modifications, providing the first steps towards analyzing the spatiotemporal variations in epigenetic regulation of cancer cell phenotypes. This study, to our knowledge, also represents the first time broad-spectrum epigenomic analysis has been applied to small cell samples collected from engineered micro-tissues.

3D Microtissue Models to Analyze the Effects of Ultralow Dose LPS on Vascular Sprouting Dynamics in the Tumor Microenvironment.

Lipopolysaccharide (LPS) plays a major role in innate immune responses and has been shown to impact vascular dynamics when present at high concentrations. However, the impact of ultralow levels of LPS (<100 pg/mL), present in the body during states of chronic inflammation, on vascular dynamics is unclear. In this study, we have integrated a 3D collagen hydrogel tissue mimic with advanced imaging and cell characterization assays to assess the potential impact of chronic inflammation on vascular dynamics, and uncover any alterations in the vascular response to low vs high dose LPS in the context of tumor progression. Accounting for both frequency of sprouting and invasiveness of the sprouts, the treatments of ultralow dose LPS with vascular endothelial growth factor (VEGF), a potent angiogenic promoter and present in excess in the tumor microenvironment, produced enhanced vascular development of human brain microvascular endothelial cells (HBMECs) in our in vitro model. There was no evidence of altered proliferation or apoptosis among the various VEGF treatment groups, indicating an enhanced migratory endothelial cell phenotype results from exposure to ultralow dose LPS with VEGF. The lack of enhanced vascular development upon treatments of high doses of LPS in the presence of VEGF could be partially attributed to an LPS dose-dependent increase in the activation of NF-κB. This study provides insight into the dynamic regulation of vascular development by varying levels of LPS and the potential role of chronic inflammation to prime a pro-angiogenic microenvironment and contribute to tumor progression.

Toward the Broad Adoption of 3D Tumor Models in the Cancer Drug Pipeline.

Despite a cost of approximately $1 billion to develop a new cancer drug, about 90% of drugs that enter clinical trials fail. A tremendous opportunity exists to streamline the drug selection and testing process, and innovative approaches promise to reduce the burdensome cost of health care for those suffering from cancer. There is great potential for 3D models of human tumors to complement more traditional testing methods; however, the shift from 2D to 3D assays at early stages of the drug discovery and development process is far from widely accepted. 3D platforms range from simple tumor spheroids to more complex microfluidic hydrogels that better mimic the tumor microenvironment. While several companies have developed and patented advanced high-throughput 3D platforms for drug screening, their cost and complexity have limited their adoption as an industry standard. In this review, we will highlight the various tumor platforms that have been developed, emphasizing the approaches that have successfully led to commercial products. We will then consider potential directions toward more relevant tumor models, advantages of the adoption of such platforms within the drug development and screening process, and new opportunities in personalized medicine that such platforms will uniquely enable.