Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models.

The lack of inadequate preclinical models remains a limitation for cancer drug development and is a primary contributor to anti-cancer drug failures in clinical trials. Heterotypic multicellular spheroids are three-dimensional (3D) spherical structures generated by self-assembly from aggregates of two or more cell types. Compared to traditional monolayer cell culture models, the organization of cells into a 3D tissue-like structure favors relevant physiological conditions with chemical and physical gradients as well as cell-cell and cell-extracellular matrix (ECM) interactions that recapitulate many of the hallmarks of cancer in situ. Epidermal growth factor receptor (EGFR) mutations are prevalent in non-small cell lung cancer (NSCLC), yet various mechanisms of acquired resistance, including epithelial-to-mesenchymal transition (EMT), limit the clinical benefit of EGFR tyrosine kinase inhibitors (EGFRi). Improved preclinical models that incorporate the complexity induced by epithelial-to-mesenchymal plasticity (EMP) are urgently needed to advance new therapeutics for clinical NSCLC management. This study was designed to provide a thorough characterization of multicellular spheroids of isogenic cancer cells of various phenotypes and demonstrate proof-of-principle for the applicability of the presented spheroid model to evaluate the impact of cancer cell phenotype in drug screening experiments through high-dimensional and spatially resolved imaging mass cytometry (IMC) analyses. First, we developed and characterized 3D homotypic and heterotypic spheroid models comprising EGFRi-sensitive or EGFRi-resistant NSCLC cells. We observed that the degree of EMT correlated with the spheroid generation efficiency in monocultures. In-depth characterization of the multicellular heterotypic spheroids using immunohistochemistry and high-dimensional single-cell analyses by IMC revealed intrinsic differences between epithelial and mesenchymal-like cancer cells with respect to self-sorting, spatiotemporal organization, and stromal cell interactions when co-cultured with fibroblasts. While the carcinoma cells harboring an epithelial phenotype self-organized into a barrier sheet surrounding the fibroblasts, mesenchymal-like carcinoma cells localized to the central hypoxic and collagen-rich areas of the compact heterotypic spheroids. Further, deep-learning-based single-cell segmentation of IMC images and application of dimensionality reduction algorithms allowed a detailed visualization and multiparametric analysis of marker expression across the different cell subsets. We observed a high level of heterogeneity in the expression of EMT markers in both the carcinoma cell populations and the fibroblasts. Our study supports further application of these models in pre-clinical drug testing combined with complementary high-dimensional single-cell analyses, which in turn can advance our understanding of the impact of cancer-stroma interactions and epithelial phenotypic plasticity on innate and acquired therapy resistance in NSCLC.

Metabolic flux analysis of 3D spheroids reveals significant differences in glucose metabolism from matched 2D cultures of colorectal cancer and pancreatic ductal adenocarcinoma cell lines.

BACKGROUND: Most in vitro cancer cell experiments have been performed using 2D models. However, 3D spheroid cultures are increasingly favored for being more representative of in vivo tumor conditions. To overcome the translational challenges with 2D cell cultures, 3D systems better model more complex cell-to-cell contact and nutrient levels present in a tumor, improving our understanding of cancer complexity. Despite this need, there are few reports on how 3D cultures differ metabolically from 2D cultures. METHODS: Well-described cell lines from colorectal cancer (HCT116 and SW948) and pancreatic ductal adenocarcinoma (Panc-1 and MIA-Pa-Ca-2) were used to investigate metabolism in 3D spheroid models. The metabolic variation under normal glucose conditions were investigated comparing 2D and 3D cultures by metabolic flux analysis and expression of key metabolic proteins. RESULTS: We find significant differences in glucose metabolism of 3D cultures compared to 2D cultures, both related to glycolysis and oxidative phosphorylation. Spheroids have higher ATP-linked respiration in standard nutrient conditions and higher non-aerobic ATP production in the absence of supplemented glucose. In addition, ATP-linked respiration is significantly inversely correlated with OCR/ECAR (p = 0.0096). Mitochondrial transport protein, TOMM20, expression decreases in all spheroid models compared to 2D, and monocarboxylate transporter (MCT) expression increases in 3 of the 4 spheroid models. CONCLUSIONS: In this study of CRC and PDAC cell lines, we demonstrate that glucose metabolism in 3D spheroids differs significantly from 2D cultures, both in terms of glycolytic and oxidative phosphorylation metrics. The metabolic phenotype shift from 2D to 3D culture in one cell line is greater than the phenotypic differences between each cell line and tumor source. The results herein emphasize the need to use 3D cell models for investigating nutrient utilization and metabolic flux for a better understanding of tumor metabolism and potential metabolic therapeutic targets.

Metformin treatment response is dependent on glucose growth conditions and metabolic phenotype in colorectal cancer cells.

Cancer cells exhibit altered metabolism, a phenomenon described a century ago by Otto Warburg. However, metabolic drug targeting is considered an underutilized and poorly understood area of cancer therapy. Metformin, a metabolic drug commonly used to treat type 2 diabetes, has been associated with lower cancer incidence, although studies are inconclusive concerning effectiveness of the drug in treatment or cancer prevention. The aim of this study was to determine how glucose concentration influences cancer cells' response to metformin, highlighting why metformin studies are inconsistent. We used two colorectal cancer cell lines with different growth rates and clinically achievable metformin concentrations. We found that fast growing SW948 are more glycolytic in terms of metabolism, while the slower growing SW1116 are reliant on mitochondrial respiration. Both cell lines show inhibitory growth after metformin treatment under physiological glucose conditions, but not in high glucose conditions. Furthermore, SW1116 converges with SW948 at a more glycolytic phenotype after metformin treatment. This metabolic shift is supported by changed GLUT1 expression. Thus, cells having different metabolic phenotypes, show a clear differential response to metformin treatment based on glucose concentration. This demonstrates the importance of growth conditions for experiments or clinical studies involving metabolic drugs such as metformin.

Blocking Aerobic Glycolysis by Targeting Pyruvate Dehydrogenase Kinase in Combination with EGFR TKI and Ionizing Radiation Increases Therapeutic Effect in Non-Small Cell Lung Cancer Cells.

Increased glycolytic activity is a hallmark of cancer initiation and progression and is often observed in non-small cell lung cancer (NSCLC). Pyruvate dehydrogenase (PDH) complex acts as a gatekeeper between glycolysis and oxidative phosphorylation, and activation of PDH is known to inhibit glycolytic activity. As part of a standard therapeutic regimen, patients with NSCLC harboring oncogenic mutations in the epidermal growth factor receptor (EGFR) are treated with EGFR tyrosine kinase inhibitors (EGFR TKIs). Independent of good initial response, development of resistance to this therapy is inevitable. In the presented work, we propose that inhibition of glycolysis will add to the therapeutic effects and possibly prevent development of resistance against both EGFR TKIs and ionizing radiation in NSCLC. Analysis of transcriptome data from two independent NSCLC patient cohorts identified increased expression of pyruvate dehydrogenase kinase 1 (PDHK1) as well as upregulated expression of genes involved in glucose metabolism in tumors compared to normal tissue. We established in vitro models of development of resistance to EGFR TKIs to study metabolism and determine if targeting PDHK would prevent development of resistance to EGFR TKIs in NSCLC cells. The PDHK1 inhibitor dichloroacetate (DCA) in combination with EGFR TKIs and/or ionizing radiation was shown to increase the therapeutic effect in our NSCLC cell models. This mechanism was associated with redirected metabolism towards pyruvate oxidation and reduced lactate production, both in EGFR TKI sensitive and resistant NSCLC cells. Using DCA, the intracellular pool of pyruvate available for lactic fermentation becomes limited. Consequently, pyruvate is redirected to the mitochondria, and reinforces mitochondrial activity. Addition of DCA to cell culture deacidifies the extracellular microenvironment as less lactate is produced and excreted. In our study, we find that this redirection of metabolism adds to the therapeutic effect of EGFR TKI and ionizing radiation in NSCLC.

AXL Targeting Abrogates Autophagic Flux and Induces Immunogenic Cell Death in Drug-Resistant Cancer Cells.

INTRODUCTION: Acquired cancer therapy resistance evolves under selection pressure of immune surveillance and favors mechanisms that promote drug resistance through cell survival and immune evasion. AXL receptor tyrosine kinase is a mediator of cancer cell phenotypic plasticity and suppression of tumor immunity, and AXL expression is associated with drug resistance and diminished long-term survival in a wide range of malignancies, including NSCLC. METHODS: We aimed to investigate the mechanisms underlying AXL-mediated acquired resistance to first- and third-generation small molecule EGFR tyrosine kinase inhibitors (EGFRi) in NSCLC. RESULTS: We found that EGFRi resistance was mediated by up-regulation of AXL, and targeting AXL reduced reactivation of the MAPK pathway and blocked onset of acquired resistance to long-term EGFRi treatment in vivo. AXL-expressing EGFRi-resistant cells revealed phenotypic and cell signaling heterogeneity incompatible with a simple bypass signaling mechanism, and were characterized by an increased autophagic flux. AXL kinase inhibition by the small molecule inhibitor bemcentinib or siRNA mediated AXL gene silencing was reported to inhibit the autophagic flux in vitro, bemcentinib treatment blocked clonogenicity and induced immunogenic cell death in drug-resistant NSCLC in vitro, and abrogated the transcription of autophagy-associated genes in vivo. Furthermore, we found a positive correlation between AXL expression and autophagy-associated gene signatures in a large cohort of human NSCLC (n = 1018). CONCLUSION: Our results indicate that AXL signaling supports a drug-resistant persister cell phenotype through a novel autophagy-dependent mechanism and reveals a unique immunogenic effect of AXL inhibition on drug-resistant NSCLC cells.

The homeobox factor Irx3 maintains adipogenic identity.

BACKGROUND: Inhibition of Irx3 and Irx5 has been shown to reduce body weight and white adipose tissue (WAT) mass through cell-autonomous and sympathetic-induced increases in adipocyte beiging and thermogenesis in mice and humans. However, the underlying mechanisms of the Irx control over beiging are still largely unknown, as illustrated by recent reports showing divergent effects of Irx3 on adipocyte metabolism and function. Here, we investigated the role of Irx3 in controlling beige preadipocyte function and differentiation. METHODS: Stable knock out of Irx3 in ME3 mouse preadipocytes capable of beiging was performed using a CRISPR-Cas9 system, and the effect on cell differentiation was assessed by qPCR, RNA-seq, Oil-red-O lipid staining and Alcian Blue staining of proteoglycans. Changes in cell identities were validated using cell type enrichment analysis from RNA-seq data. Proliferation and cell cycle progression in undifferentiated cells were measured by WST-1 and flow cytometry, reactive oxygen species (ROS) generation was determined by fluorescence spectrometry and mitochondrial respiration was investigated by Seahorse assay. RESULTS: Irx3 was found to be essential for the identity, function and adipogenic differentiation of beige adipocyte precursors. Irx3-KO impaired proliferation, ROS generation and mitochondrial respiration in the preadipocytes. We further observed profound changes in numerous genes during both early and late stages of adipogenic differentiation, including genes important for adipocyte differentiation, cell cycle progression, oxidative phosphorylation (OXPHOS) and morphogenesis. Irx3-KO cells failed to accumulate lipids following adipogenic stimuli, and cell enrichment analysis revealed a loss of preadipocyte identity and a gain of chondrocyte-like identity in Irx3-KO cells during early differentiation. Finally, unlike the control cells, the Irx3-KO cells readily responded to chondrogenic stimuli. CONCLUSIONS: Irx3 is required for preadipocyte identity and differentiation capacity. Our findings suggest that, while inhibition of Irx3 may be beneficial during later developmental stages to modulate adipogenesis in the beige direction, constitutive and complete absence of Irx3 in the embryonic fibroblast stage leads to detrimental loss of adipogenic differentiation capacity.

Epithelial to mesenchymal transition (EMT) is associated with attenuation of succinate dehydrogenase (SDH) in breast cancer through reduced expression of SDHC.

BACKGROUND: Epithelial to mesenchymal transition (EMT) is a well-characterized process of cell plasticity that may involve metabolic rewiring. In cancer, EMT is associated with malignant progression, tumor heterogeneity, and therapy resistance. In this study, we investigated the role of succinate dehydrogenase (SDH) as a potential key regulator of EMT. METHODS: Associations between SDH subunits and EMT were explored in gene expression data from breast cancer patient cohorts, followed by in-depth studies of SDH suppression as a potential mediator of EMT in cultured cells. RESULTS: We found an overall inverse association between EMT and the SDH subunit C (SDHC) when analyzing gene expression in breast tumors. This was particularly evident in carcinomas of basal-like molecular subtype compared to non-basal-like tumors, and a low SDHC expression level tended to have a prognostic impact in those patients. Studies in cultured cells revealed that EMT was induced by SDH inhibition through SDHC CRISPR/Cas9 knockdown or by the enzymatic inhibitor malonate. Conversely, overexpression of EMT-promoting transcription factors TWIST and SNAI2 caused decreased levels of SDHB and C and reduced rates of SDH-linked mitochondrial respiration. Cells overexpressing TWIST had reduced mitochondrial mass, and the organelles were thinner and more fragmented compared to controls. CONCLUSIONS: Our findings suggest that downregulation of SDHC promotes EMT and that this is accompanied by structural remodeling of the mitochondrial organelles. This may confer survival benefits upon exposure to hostile microenvironment including oxidative stress and hypoxia during cancer progression.

Introducing nano-scale quantitative polymerase chain reaction.

We show that the quantitative PCR reaction volume can be reduced significantly compared to the standard procedures, without compromising data quality. By analyzing dilution series (100-10-7) we found that measurement in 1 µl reaction volume indeed gave valid results comparable to 10 µL, when using a routine pipetting robot. This may enable a significant cost reduction through cutback of both biological material as well as chemical reagents.

EGFR heterogeneity and implications for therapeutic intervention in glioblastoma.

Patients with glioblastoma (GBM) have a universally poor prognosis and are in urgent need of effective treatment strategies. Recent advances in sequencing techniques unraveled the complete genomic landscape of GBMs and revealed profound heterogeneity of individual tumors even at the single cell level. Genomic profiling has detected epidermal growth factor receptor (EGFR) gene alterations in more than half of GBMs. Major genetic events include amplification and mutation of EGFR. Yet, treatment strategies targeting EGFR have thus far failed in clinical trials. In this review, we discuss the clonal and functional heterogeneity of EGFRs in GBM development and critically reassess the potential of EGFRs as therapeutic targets.

The angiogenic switch leads to a metabolic shift in human glioblastoma.

Background: Invasion and angiogenesis are major hallmarks of glioblastoma (GBM) growth. While invasive tumor cells grow adjacent to blood vessels in normal brain tissue, tumor cells within neovascularized regions exhibit hypoxic stress and promote angiogenesis. The distinct microenvironments likely differentially affect metabolic processes within the tumor cells. Methods: In the present study, we analyzed gene expression and metabolic changes in a human GBM xenograft model that displayed invasive and angiogenic phenotypes. In addition, we used glioma patient biopsies to confirm the results from the xenograft model. Results: We demonstrate that the angiogenic switch in our xenograft model is linked to a proneural-to-mesenchymal transition that is associated with upregulation of the transcription factors BHLHE40, CEBPB, and STAT3. Metabolic analyses revealed that angiogenic xenografts employed higher rates of glycolysis compared with invasive xenografts. Likewise, patient biopsies exhibited higher expression of the glycolytic enzyme lactate dehydrogenase A and glucose transporter 1 in hypoxic areas compared with the invasive edge and lower-grade tumors. Analysis of the mitochondrial respiratory chain showed reduction of complex I in angiogenic xenografts and hypoxic regions of GBM samples compared with invasive xenografts, nonhypoxic GBM regions, and lower-grade tumors. In vitro hypoxia experiments additionally revealed metabolic adaptation of invasive tumor cells, which increased lactate production under long-term hypoxia. Conclusions: The use of glycolysis versus mitochondrial respiration for energy production within human GBM tumors is highly dependent on the specific microenvironment. The metabolic adaptability of GBM cells highlights the difficulty of targeting one specific metabolic pathway for effective therapeutic intervention.

Metabolic profiling indicates impaired pyruvate dehydrogenase function in myalgic encephalopathy/chronic fatigue syndrome.

Myalgic encephalopathy/chronic fatigue syndrome (ME/CFS) is a debilitating disease of unknown etiology, with hallmark symptoms including postexertional malaise and poor recovery. Metabolic dysfunction is a plausible contributing factor. We hypothesized that changes in serum amino acids may disclose specific defects in energy metabolism in ME/CFS. Analysis in 200 ME/CFS patients and 102 healthy individuals showed a specific reduction of amino acids that fuel oxidative metabolism via the TCA cycle, mainly in female ME/CFS patients. Serum 3-methylhistidine, a marker of endogenous protein catabolism, was significantly increased in male patients. The amino acid pattern suggested functional impairment of pyruvate dehydrogenase (PDH), supported by increased mRNA expression of the inhibitory PDH kinases 1, 2, and 4; sirtuin 4; and PPARδ in peripheral blood mononuclear cells from both sexes. Myoblasts grown in presence of serum from patients with severe ME/CFS showed metabolic adaptations, including increased mitochondrial respiration and excessive lactate secretion. The amino acid changes could not be explained by symptom severity, disease duration, age, BMI, or physical activity level among patients. These findings are in agreement with the clinical disease presentation of ME/CFS, with inadequate ATP generation by oxidative phosphorylation and excessive lactate generation upon exertion.

EGFRvIII mutations can emerge as late and heterogenous events in glioblastoma development and promote angiogenesis through Src activation.

BACKGROUND: Amplification of the epidermal growth factor receptor (EGFR) and its mutant EGFRvIII are among the most common genetic alterations in glioblastoma (GBM), the most frequent and most aggressive primary brain tumor. METHODS: In the present work, we analyzed the clonal evolution of these major EGFR aberrations in a small cohort of GBM patients using a unique surgical multisampling technique. Furthermore, we overexpressed both receptors separately and together in 2 patient-derived GBM stem cell lines (GSCs) to analyze their functions in vivo in orthotopic xenograft models. RESULTS: In human GBM biopsies, we identified EGFR amplification as an early event because EGFRvIII mutations emerge from intratumoral heterogeneity later in tumor development. To investigate the biological relevance of this distinct developmental pattern, we established experimental model systems. In these models, EGFR+ tumor cells showed activation of classical downstream signaling pathways upon EGF stimulation and displayed enhanced invasive growth without evidence of angiogenesis in vivo. In contrast, EGFRvIII+ tumors were driven by activation of the prototypical Src family kinase c-Src that promoted VEGF secretion leading to angiogenic tumor growth. CONCLUSIONS: The presented work shows that sequential EGFR amplification and EGFRvIII mutations might represent concerted evolutionary events that drive the aggressive nature of GBM by promoting invasion and angiogenesis via distinct signaling pathways. In particular, c-SRC may be an attractive therapeutic target for tumors harboring EGFRvIII as we identified this protein specifically mediating angiogenic tumor growth downstream of EGFRvIII.

Tumor versus stromal cells in culture--survival of the fittest?

Two of the signature genetic events that occur in human gliomas, EGFR amplification and IDH mutation, are poorly represented in experimental models in vitro. EGFR amplification, for example, occurs in 40 to 50% of GBM, and yet, EGFR amplification is rarely preserved in cell cultures derived from human tumors. To analyze the fate of EGFR amplified and IDH mutated cells in culture, we followed the development over time of cultures derived from human xenografts in nude rats enriched for tumor cells with EGFR amplification and of cultures derived from patient samples with IDH mutations, in serum monolayer and spheroid suspension culture, under serum and serum free conditions. We observed under serum monolayer conditions, that nestin positive or nestin and SMA double positive rat stromal cells outgrew EGFR amplified tumor cells, while serum spheroid cultures preserved tumor cells with EGFR amplification. Serum free suspension culture exhibited a more variable cell composition in that the resultant cell populations were either predominantly nestin/SOX2 co-expressing rat stromal cells or human tumor cells, or a mixture of both. The selection for nestin/SMA positive stromal cells under serum monolayer conditions was also consistently observed in human oligodendrogliomas and oligoastrocytomas with IDH mutations. Our results highlight for the first time that serum monolayer conditions can select for stromal cells instead of tumor cells in certain brain tumor subtypes. This result has an important impact on the establishment of new tumor cell cultures from brain tumors and raises the question of the proper conditions for the growth of the tumor cell populations of interest.

EGFR wild-type amplification and activation promote invasion and development of glioblastoma independent of angiogenesis.

Angiogenesis is regarded as a hallmark of cancer progression and it has been postulated that solid tumor growth depends on angiogenesis. At present, however, it is clear that tumor cell invasion can occur without angiogenesis, a phenomenon that is particularly evident by the infiltrative growth of malignant brain tumors, such as glioblastomas (GBMs). In these tumors, amplification or overexpression of wild-type (wt) or truncated and constitutively activated epidermal growth factor receptor (EGFR) are regarded as important events in GBM development, where the complex downstream signaling events have been implicated in tumor cell invasion, angiogenesis and proliferation. Here, we show that amplification and in particular activation of wild-type EGFR represents an underlying mechanism for non-angiogenic, invasive tumor growth. Using a clinically relevant human GBM xenograft model, we show that tumor cells with EGFR gene amplification and activation diffusely infiltrate normal brain tissue independent of angiogenesis and that transient inhibition of EGFR activity by cetuximab inhibits the invasive tumor growth. Moreover, stable, long-term expression of a dominant-negative EGFR leads to a mesenchymal to epithelial-like transition and induction of angiogenic tumor growth. Analysis of human GBM biopsies confirmed that EGFR activation correlated with invasive/non-angiogenic tumor growth. In conclusion, our results indicate that activation of wild-type EGFR promotes invasion and glioblastoma development independent of angiogenesis, whereas loss of its activity results in angiogenic tumor growth.

Comment to: "Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro" by Z. Ren et al. Exp. Cell Res. 317 (2011) 2950-2957: spontaneous transformation of mesenchymal stem cells in culture: facts or fiction?

There is at present a controversy in the literature whether MSCs are susceptible to spontaneous in vitro transformation or not. Several groups have reported spontaneous transformation of MSCs from various species. However, some of these reports were not true transformations and later proven to be due to cross-contaminating cancer cells. To date there is no solid evidence that MSCs can undergo spontaneous transformation in culture. Only two groups used DNA fingerprinting to authenticate their transformed cells, and both groups later showed cross-contamination of cancer cells in their cultures. In this commentary, we address the paper "Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro" by Z. Ren et al. Exp. Cell Res. 317 (2011) 2950-2957. In this article the authors characterize the transformed mesenchymal cells (TMCs) and claim to have verified their origin. We question the authentication of the TMCs made by the authors and we also believe it is in the interest of the scientific community, that a highly controversial finding, such as spontaneous transformation of MSCs, should be properly verified by stringent methods, preferably DNA fingerprinting, in order to validate if an actual transformation event has occurred.