Three-Dimensional Tumor Models to Study Cancer Stemness-Mediated Drug Resistance.

Solid tumors often contain genetically different populations of cancer cells, stromal cells, various structural and soluble proteins, and other soluble signaling molecules. The American Cancer society estimated 1,958,310 new cancer cases and 609,820 cancer deaths in the United States in 2023. A major barrier against successful treatment of cancer patients is drug resistance. Gain of stem cell-like states by cancer cells under drug pressure or due to interactions with the tumor microenvironment is a major mechanism that renders therapies ineffective. Identifying approaches to target cancer stem cells is expected to improve treatment outcomes for patients. Most of our understanding of drug resistance and the role of cancer stemness is from monolayer cell cultures. Recent advances in cell culture technologies have enabled developing sophisticated three-dimensional tumor models that facilitate mechanistic studies of cancer drug resistance. This review summarizes the role of cancer stemness in drug resistance and highlights the various tumor models that are used to discover the underlying mechanisms and test potentially novel therapeutics.

Comparison of Engineered Liver 3D Models and the Role of Oxygenation for Patient-Derived Tumor Cells and Immortalized Cell Lines Cocultured with Tumor Stroma in the Detection of Hepatotoxins.

In metabolically active tumors, responses of cells to drugs are heavily influenced by oxygen availability via the surrounding vasculature alongside the extracellular matrix signaling. The objective of this study is to investigate hepatotoxicity by replicating critical features of hepatocellular carcinoma (HCC). This includes replicating 3D structures, metabolic activities, and tumor-specific markers. The internal environment of spheroids comprised of cancerous human patient-derived hepatocytes using microparticles is modulated to enhance the oxygenation state and recreate cell-extracellular matrix interactions. Furthermore, the role of hepatic stellate cells in maintaining hepatocyte survival and function is explored and hepatocytes from two cellular sources (immortalized and patient-derived) to create four formulations with and without microparticles are utilized. To investigate drug-induced changes in metabolism and apoptosis in liver cells, coculture spheroids with and without microparticles are exposed to three hepatotoxic drugs. The use of microparticles increases levels of apoptotic markers in both liver models under drug treatments. This coincides with reduced levels of anti-apoptotic proteins and increased levels of pro-apoptotic proteins. Moreover, cells from different origins undergo apoptosis through distinct apoptotic pathways in response to identical drugs. This 3D microphysiological system offers a viable tool for liver cancer research to investigate mechanisms of apoptosis under different microenvironmental conditions.

Inhibiting BRAF/EGFR/MEK suppresses cancer stemness and drug resistance of primary colorectal cancer cells.

Drug resistance is a major barrier against successful treatments of cancer patients. Gain of stemness under drug pressure is a major mechanism that renders treatments ineffective. Identifying approaches to target cancer stem cells (CSCs) is expected to improve treatment outcomes for patients. To elucidate the role of cancer stemness in resistance of colorectal cancer cells to targeted therapies, we developed spheroid cultures of patient-derived BRAFmut and KRASmut tumor cells and studied resistance mechanisms to inhibition of MAPK pathway through phenotypic and gene and protein expression analysis. We found that treatments enriched the expression of CSC markers CD166, ALDH1A3, CD133, and LGR5 and activated PI3K/Akt pathway in cancer cells. We examined various combination treatments to block these activities and found that a triple combination against BRAF, EGFR, and MEK significantly reduced stemness and activities of oncogenic signaling pathways. This study demonstrates the feasibility of blocking stemness-mediated drug resistance and tumorigenic activities in colorectal cancer.

Therapeutic Targeting of Cancer Stem Cells Prevents Resistance of Colorectal Cancer Cells to MEK Inhibition.

Drug resistance is a leading cause for the failure of cancer treatments. Plasticity of cancer cells to acquire stem cell-like properties enables them to escape drug toxicity through different adaptive mechanisms. Eliminating cancer stem cells (CSCs) can potentially improve treatment outcomes for patients. To determine the role of CSCs in resistance of colorectal cancer cells to targeted therapies and identify treatment strategies, we treated spheroids of BRAFmut and KRASmut colorectal cancer cells with inhibitors of the mitogen-activated protein kinase pathway and studied resistance mechanisms through gene and protein expression analyses. We found that treatments activated several oncogenic pathways and expression of CSC markers CD166 and ALDH1A3. We identified a specific combination treatment using trametinib and mithramycin A to simultaneously inhibit the CSC phenotype and activities of several pathways in cancer cells. This study demonstrates the feasibility of therapeutic targeting of CSCs as a strategy to block tumorigenic activities of cancer cells.

Therapeutic Targeting of Stromal-Tumor HGF-MET Signaling in an Organotypic Triple-Negative Breast Tumor Model.

The tumor microenvironment (TME) promotes proliferation, drug resistance, and invasiveness of cancer cells. Therapeutic targeting of the TME is an attractive strategy to improve outcomes for patients, particularly in aggressive cancers such as triple-negative breast cancer (TNBC) that have a rich stroma and limited targeted therapies. However, lack of preclinical human tumor models for mechanistic understanding of tumor-stromal interactions has been an impediment to identify effective treatments against the TME. To address this need, we developed a three-dimensional organotypic tumor model to study interactions of patient-derived cancer-associated fibroblasts (CAF) with TNBC cells and explore potential therapy targets. We found that CAFs predominantly secreted hepatocyte growth factor (HGF) and activated MET receptor tyrosine kinase in TNBC cells. This tumor-stromal interaction promoted invasiveness, epithelial-to-mesenchymal transition, and activities of multiple oncogenic pathways in TNBC cells. Importantly, we established that TNBC cells become resistant to monotherapy and demonstrated a design-driven approach to select drug combinations that effectively inhibit prometastatic functions of TNBC cells. Our study also showed that HGF from lung fibroblasts promotes colony formation by TNBC cells, suggesting that blocking HGF-MET signaling potentially could target both primary TNBC tumorigenesis and lung metastasis. Overall, we established the utility of our organotypic tumor model to identify and therapeutically target specific mechanisms of tumor-stromal interactions in TNBC toward the goal of developing targeted therapies against the TME. IMPLICATIONS: Leveraging a state-of-the-art organotypic tumor model, we demonstrated that CAFs-mediated HGF-MET signaling drive tumorigenic activities in TNBC and presents a therapeutic target.

Modeling adaptive drug resistance of colorectal cancer and therapeutic interventions with tumor spheroids.

Drug resistance is a major barrier against successful treatments of cancer patients. Various intrinsic mechanisms and adaptive responses of tumor cells to cancer drugs often lead to failure of treatments and tumor relapse. Understanding mechanisms of cancer drug resistance is critical to develop effective treatments with sustained anti-tumor effects. Three-dimensional cultures of cancer cells known as spheroids present a biologically relevant model of avascular tumors and have been increasingly incorporated in tumor biology and cancer drug discovery studies. In this review, we discuss several recent studies from our group that utilized colorectal tumor spheroids to investigate responses of cancer cells to cytotoxic and molecularly targeted drugs and uncover mechanisms of drug resistance. We highlight our findings from both short-term, one-time treatments and long-term, cyclic treatments of tumor spheroids and discuss mechanisms of adaptation of cancer cells to the treatments. Guided by mechanisms of resistance, we demonstrate the feasibility of designing specific drug combinations to effectively block growth and resistance of cancer cells in spheroid cultures. Finally, we conclude with our perspectives on the utility of three-dimensional tumor models and their shortcomings and advantages for phenotypic and mechanistic studies of cancer drug resistance.

Toxicity of Combinations of Kinase Pathway Inhibitors to Normal Human Cells in a Three-Dimensional Culture.

Resistance to single-agent chemotherapy and molecularly targeted drugs prevents sustained efficacy of treatments. To address this challenge, combination drug treatments have been used to improve outcomes for patients. Potential toxicity of combination treatments is a major concern, however, and has led to the failure of several clinical trials in different cancers. The use of cell-based models of normal tissues in preclinical studies enables testing and identifying toxic effects of drug combinations and facilitates an informed decision-making process for advancing the treatments to animal models and clinical trials. Recently, we established that combinations of molecular inhibitors of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase-protein kinase B (PI3K/Akt) pathways effectively and synergistically inhibit growth of BRAFmut and KRASmut colorectal tumor spheroids by blocking feedback signaling of downstream kinase pathways. These pathways are important for cell proliferation, however, and their simultaneous inhibition may cause toxicity to normal cells. We used a cellular spheroid model to study toxicities of drug combinations to human bone marrow and colon. Our results indicated that MAPK and PI3K/Akt inhibitors used simultaneously were only moderately toxic to bone marrow cells but significantly more toxic to colon cells. Our molecular analysis of proliferative cell activities and housekeeping proteins further corroborated these results. Overall, our approach to identify toxic effects of combinations of cancer drugs to normal cells in three-dimensional cultures will facilitate more informed treatment selections for subsequent animal studies.

Modeling Adaptive Resistance of KRAS Mutant Colorectal Cancer to MAPK Pathway Inhibitors with a Three-Dimensional Tumor Model.

Single-agent drug treatment of KRASmut colorectal cancers is often ineffective because the activation of compensatory signaling pathways leads to drug resistance. To mimic cyclic chemotherapy treatments of patients, we showed that intermittent treatments of 3D tumor spheroids of KRASmut colorectal cancer cells with inhibitors of mitogen-activated protein kinase (MAPK) signaling pathway temporarily suppressed growth of spheroids. However, the efficacy of successive single-agent treatments was significantly reduced. Molecular analysis showed compensatory activation of PI3K/AKT and STAT kinases and EGFR family proteins. To overcome the adaptation of cancer cells to MAPK pathway inhibitors, we treated tumor spheroids with a combination of MEK and EGFR inhibitors. This approach significantly blocked signaling of MAPK and PI3K/AKT pathways and prevented the growth of spheroids, but it was not effective against STAT signaling. Although the combination treatment blocked the matrix invasion of DLD1 cells, additional treatments with STAT inhibitors were necessary to prevent invasiveness of HCT116 cells. Overall, our drug resistance model elucidated the mechanisms of treatment-induced growth and invasiveness of cancer cells and allowed design-driven testing and identifying of effective treatments to suppress these phenotypes.