Dissecting the Role of AXL in Cancer Immune Escape and Resistance to Immune Checkpoint Inhibition.

The development and implementation of Immune Checkpoint Inhibitors (ICI) in clinical oncology have significantly improved the survival of a subset of cancer patients with metastatic disease previously considered uniformly lethal. However, the low response rates and the low number of patients with durable clinical responses remain major concerns and underscore the limited understanding of mechanisms regulating anti-tumor immunity and tumor immune resistance. There is an urgent unmet need for novel approaches to enhance the efficacy of ICI in the clinic, and for predictive tools that can accurately predict ICI responders based on the composition of their tumor microenvironment. The receptor tyrosine kinase (RTK) AXL has been associated with poor prognosis in numerous malignancies and the emergence of therapy resistance. AXL is a member of the TYRO3-AXL-MERTK (TAM) kinase family. Upon binding to its ligand GAS6, AXL regulates cell signaling cascades and cellular communication between various components of the tumor microenvironment, including cancer cells, endothelial cells, and immune cells. Converging evidence points to AXL as an attractive molecular target to overcome therapy resistance and immunosuppression, supported by the potential of AXL inhibitors to improve ICI efficacy. Here, we review the current literature on the prominent role of AXL in regulating cancer progression, with particular attention to its effects on anti-tumor immune response and resistance to ICI. We discuss future directions with the aim to understand better the complex role of AXL and TAM receptors in cancer and the potential value of this knowledge and targeted inhibition for the benefit of cancer patients.

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.

Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to the initiation of unprecedented research efforts to understand the pathogenesis mediated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). More knowledge is needed regarding the cell type-specific cytopathology and its impact on cellular tropism. Furthermore, the impact of novel SARS-CoV-2 mutations on cellular tropism, alternative routes of entry, the impact of co-infections, and virus replication kinetics along the respiratory tract remains to be explored in improved models. Most applied virology models are not well suited to address the remaining questions, as they do not recapitulate the histoarchitecture and cellular composition of human respiratory tissues. The overall aim of this work was to establish from single biopsy specimens, a human adult stem cell-derived organoid model representing the upper respiratory airways and lungs and explore the applicability of this model to study respiratory virus infection. First, we characterized the organoid model with respect to growth pattern and histoarchitecture, cellular composition, and functional characteristics. Next, in situ expression of viral entry receptors, including influenza virus-relevant sialic acids and SARS-CoV-2 entry receptor ACE2 and TMPRSS2, were confirmed in organoids of bronchiolar and alveolar differentiation. We further showed successful infection by pseudotype influenza A H7N1 and H5N1 virus, and the ability of the model to support viral replication of influenza A H7N1 virus. Finally, successful infection and replication of a clinical isolate of SARS-CoV-2 were confirmed in the organoids by TCID50 assay and immunostaining to detect intracellular SARS-CoV-2 specific nucleocapsid and dsRNA. The prominent syncytia formation in organoid tissues following SARS-CoV-2 infection mimics the findings from infected human tissues in situ. We conclude that the human organotypic model described here may be particularly useful for virology studies to evaluate regional differences in the host response to infection. The model contains the various cell types along the respiratory tract, expresses respiratory virus entry factors, and supports successful infection and replication of influenza virus and SARS-CoV-2. Thus, the model may serve as a relevant and reliable tool in virology and aid in pandemic preparedness, and efficient evaluation of antiviral strategies.

Bortezomib abrogates temozolomide-induced autophagic flux through an ATG5 dependent pathway.

Introduction: Glioblastoma (GBM) is invariably resistant to temozolomide (TMZ) chemotherapy. Inhibiting the proteasomal pathway is an emerging strategy to accumulate damaged proteins and inhibit their lysosomal degradation. We hypothesized that pre-treatment of glioblastoma with bortezomib (BTZ) might sensitize glioblastoma to temozolomide by abolishing autophagy survival signals to augment DNA damage and apoptosis. Methods: P3 patient-derived glioblastoma cells, as well as the tumour cell lines U87, HF66, A172, and T98G were investigated for clonogenic survival after single or combined treatment with temozolomide and bortezomib in vitro. We investigated the requirement of functional autophagy machinery by utilizing pharmacological inhibitors or CRISPR-Cas9 knockout (KO) of autophagy-related genes -5 and -7 (ATG5 and ATG7) in glioblastoma cells and monitored changes in autophagic flux after temozolomide and/or bortezomib treatments. P3 wild-type and P3 ATG5-/- (ATG5 KO) cells were implanted orthotopically into NOD-SCID mice to assess the efficacy of bortezomib and temozolomide combination therapy with and without functional autophagy machinery. Results: The chemo-resistant glioblastoma cells increased autophagic flux during temozolomide treatment as indicated by increased degradation of long-lived proteins, diminished expression of autophagy markers LC3A/B-II and p62 (SQSTM1), increased co-localisation of LC3A/B-II with STX17, augmented and no induction of apoptosis. In contrast, bortezomib treatment abrogated autophagic flux indicated by the accumulation of LC3A/B-II and p62 (SQSTM1) positive autophagosomes that did not fuse with lysosomes and thus reduced the degradation of long-lived proteins. Bortezomib synergistically enhanced temozolomide efficacy by attenuating cell proliferation, increased DNA double-strand breaks, and apoptosis in an autophagy-dependent manner. Abolishing autophagy in ATG5 KOs reversed the bortezomib-induced toxicity, rescued glioblastoma cell death and reduced animal survival. Discussion: We conclude that bortezomib abrogates temozolomide induced autophagy flux through an ATG5 dependent pathway.

Induction of alveolar and bronchiolar phenotypes in human lung organoids.

Patient-derived organoids have revolutionized biomedical research and therapies by "transferring the patient into the Petri dish". In vitro access to human lung organoids representing distal lung tissue, i.e. alveolar organoids, would facilitate research pertaining to a wide range of medical conditions and might open for a future approach to individualized treatment.We propose a protocol to derive a single human lung biopsy towards both alveolar and bronchiolar organoids. By modulating Wnt pathway, we obtained a differential gene expression of the main markers for both subtypes, such as a higher expression of surfactant protein C in alveolar organoids or a higher expression of mucine 5AC in bronchiolar organoids. Although the specific cell enrichment was not complete, the differentiation was observed as early as passage 1 based on morphology, and confirmed by QPCR and histology at passage 2. These results are consistent with a functional specification of lung epithelium towards both alveoli- and bronchi-enriched organoids from first passages.

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.

Role of Hypoxia-Mediated Autophagy in Tumor Cell Death and Survival.

Programmed cell death or type I apoptosis has been extensively studied and its contribution to the pathogenesis of disease is well established. However, autophagy functions together with apoptosis to determine the overall fate of the cell. The cross talk between this active self-destruction process and apoptosis is quite complex and contradictory as well, but it is unquestionably decisive for cell survival or cell death. Autophagy can promote tumor suppression but also tumor growth by inducing cancer-cell development and proliferation. In this review, we will discuss how autophagy reprograms tumor cells in the context of tumor hypoxic stress. We will illustrate how autophagy acts as both a suppressor and a driver of tumorigenesis through tuning survival in a context dependent manner. We also shed light on the relationship between autophagy and immune response in this complex regulation. A better understanding of the autophagy mechanisms and pathways will undoubtedly ameliorate the design of therapeutics aimed at targeting autophagy for future cancer immunotherapies.

AXL Is a Driver of Stemness in Normal Mammary Gland and Breast Cancer.

The receptor tyrosine kinase AXL is associated with epithelial plasticity in several solid tumors including breast cancer and AXL-targeting agents are currently in clinical trials. We hypothesized that AXL is a driver of stemness traits in cancer by co-option of a regulatory function normally reserved for stem cells. AXL-expressing cells in human mammary epithelial ducts co-expressed markers associated with multipotency, and AXL inhibition abolished colony formation and self-maintenance activities while promoting terminal differentiation in vitro. Axl-null mice did not exhibit a strong developmental phenotype, but enrichment of Axl + cells was required for mouse mammary gland reconstitution upon transplantation, and Axl-null mice had reduced incidence of Wnt1-driven mammary tumors. An AXL-dependent gene signature is a feature of transcriptomes in basal breast cancers and reduced patient survival irrespective of subtype. Our interpretation is that AXL regulates access to epithelial plasticity programs in MaSCs and, when co-opted, maintains acquired stemness in breast cancer cells.

Hypoxia-driven intratumor heterogeneity and immune evasion.

While it is widely accepted that high intratumoral heterogeneity confers serious challenges in the emerging resistance and the subsequent effective therapeutic targeting of cancer, the underlying biology of intratumoral heterogeneity remains elusive. In particular, it remains to be fully elucidated how microenvironmental factors shape genetic and non-genetic heterogeneity, which in turn determine the course of tumor evolution and clinical progression. In this context, hypoxia, a hallmark of most growing cancers, characterized by decreased O2 partial pressure is a key player of the tumor microenvironment. Despite extensive data indicating that hypoxia promotes cellular metabolic adaptation, immune suppression and various steps of tumor progression via hypoxia regulated gene transcription, much less is known about the role of hypoxia in mediating therapy resistance as a driver of tumor evolution through genetic and non-genetic mechanisms. In this review, we will discuss recent evidence supporting a prominent role of hypoxia as a driver of tumor heterogeneity and highlight the multifaceted manner by which this in turn could impact cancer evolution, reprogramming and immune escape. Finally, we will discuss how detailed knowledge of the hypoxic footprint may open up new therapeutic avenues for the management of cancer.

Decoding cancer's camouflage: epithelial-mesenchymal plasticity in resistance to immune checkpoint blockade.

Epithelial-mesenchymal plasticity (EMP) of cancer cells contributes to cancer cell heterogeneity, and it is well established that EMP is a critical determinant of acquired resistance to cancer treatment modalities including radiation therapy, chemotherapy, and targeted therapies. Here, we aimed to explore how EMP contributes to cancer cell camouflage, allowing an ever-changing population of cancer cells to pass under the radar of our immune system and consequently compromise the effect of immune checkpoint blockade therapies. The ultimate clinical benefit of any combination regimen is evidenced by the sum of the drug-induced alterations observed in the variety of cellular populations composing the tumor immune microenvironment. The finely-tuned molecular crosstalk between cancer and immune cells remains to be fully elucidated, particularly for the spectrum of malignant cells along the epithelial to mesenchymal axis. High-dimensional single cell analyses of specimens collected in ongoing clinical studies is becoming a key contributor to our understanding of these interactions. This review will explore to what extent targeting EMP in combination with immune checkpoint inhibition represents a promising therapeutic avenue within the overarching strategy to reactivate a halting cancer-immunity cycle and establish a robust host immune response against cancer cells. Therapeutic strategies currently in clinical development will be discussed.

AXL Targeting Overcomes Human Lung Cancer Cell Resistance to NK- and CTL-Mediated Cytotoxicity.

Immune resistance may arise from both genetic instability and tumor heterogeneity. Microenvironmental stresses such as hypoxia and various resistance mechanisms promote carcinoma cell plasticity. AXL, a member of the TAM (Tyro3, Axl, and Mer) receptor tyrosine kinase family, is widely expressed in human cancers and increasingly recognized for its role in cell plasticity and drug resistance. To investigate mechanisms of immune resistance, we studied multiple human lung cancer clones derived from a model of hypoxia-induced tumor plasticity that exhibited mesenchymal or epithelial features. We demonstrate that AXL expression is increased in mesenchymal lung cancer clones. Expression of AXL in the cells correlated with increased cancer cell-intrinsic resistance to both natural killer (NK)- and cytotoxic T lymphocyte (CTL)-mediated killing. A small-molecule targeting AXL sensitized mesenchymal lung cancer cells to cytotoxic lymphocyte-mediated killing. Mechanistically, we showed that attenuation of AXL-dependent immune resistance involved a molecular network comprising NF-κB activation, increased ICAM1 expression, and upregulation of ULBP1 expression coupled with MAPK inhibition. Higher ICAM1 and ULBP1 tumor expression correlated with improved patient survival in two non-small cell lung cancer (NSCLC) cohorts. These results reveal an AXL-mediated immune-escape regulatory pathway, suggest AXL as a candidate biomarker for tumor resistance to NK and CTL immunity, and support AXL targeting to optimize immune response in NSCLC.

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.

A novel SRC-2-dependent regulation of epithelial-mesenchymal transition in breast cancer cells.

Steroid receptor coactivator 2 (SRC-2) is a nuclear receptor coactivator, important for the regulation of estrogen receptor alpha (ERα)-mediated transcriptional activity in breast cancer cells. However, the transcriptional role of SRC-2 in breast cancer is still ambiguous. Here we aimed to unravel a more precise transcriptional role of SRC-2 and uncover unique target genes in MCF-7 breast cancer cells, as opposed to the known oncogene SRC-3. Gene expression analyses of cells depleted of either SRC-2 or SRC-3 showed that they transcriptionally regulate mostly separate gene sets. However, individual unique gene sets were implicated in some of the same major gene ontology biological processes, such as cellular structure and development. This finding was supported by three-dimensional cell cultures, demonstrating that depletion of SRC-2 and SRC-3 changed the morphology of the cells into epithelial-like hollow acinar structures, indicating that both SRC proteins are involved in maintaining the hybrid E/M phenotype. In clinical ER-positive, HER2-negative breast cancer samples the expression of SRC-2 was negatively correlated with the expression of MCF-7-related luminal, cell cycle and cellular morphogenesis genes. Finally, elucidating SRC-2 unique transcriptional effects, we identified Lyn kinase (an EMT biomarker) to be upregulated exclusively after SRC-2 depletion. In conclusion, we show that both SRC-2 and SRC-3 are essential for the EMT in breast cancer cells, controlling different transcriptional niches.

Microenvironment-Induced Non-sporadic Expression of the AXL and cKIT Receptors Are Related to Epithelial Plasticity and Drug Resistance.

The existence of rare cancer cells that sporadically acquire drug-tolerance through epigenetic mechanisms is proposed as one mechanism that drives cancer therapy failure. Here we provide evidence that specific microenvironments impose non-sporadic expression of proteins related to epithelial plasticity and drug resistance. Microarrays of robotically printed combinatorial microenvironments of known composition were used to make cell-based functional associations between microenvironments, which were design-inspired by normal and tumor-burdened breast tissues, and cell phenotypes. We hypothesized that specific combinations of microenvironment constituents non-sporadically impose the induction of the AXL and cKIT receptor tyrosine kinase proteins, which are known to be involved in epithelial plasticity and drug-tolerance, in an isogenic human mammary epithelial cell (HMEC) malignant progression series. Dimension reduction analysis reveals type I collagen as a dominant feature, inducing expression of both markers in pre-stasis finite lifespan HMECs, and transformed non-malignant and malignant immortal cell lines. Basement membrane-associated matrix proteins, laminin-111 and type IV collagen, suppress AXL and cKIT expression in pre-stasis and non-malignant cells. However, AXL and cKIT are not suppressed by laminin-111 in malignant cells. General linear models identified key factors, osteopontin, IL-8, and type VIα3 collagen, which significantly upregulated AXL and cKIT, as well as a plasticity-related gene expression program that is often observed in stem cells and in epithelial-to-mesenchymal-transition. These factors are co-located with AXL-expressing cells in situ in normal and breast cancer tissues, and associated with resistance to paclitaxel. A greater diversity of microenvironments induced AXL and cKIT expression consistent with plasticity and drug-tolerant phenotypes in tumorigenic cells compared to normal or immortal cells, suggesting a reduced perception of microenvironment specificity in malignant cells. Microenvironment-imposed reprogramming could explain why resistant cells are seemingly persistent and rapidly adaptable to multiple classes of drugs. These results support the notion that specific microenvironments drive drug-tolerant cellular phenotypes and suggest a novel interventional avenue for preventing acquired therapy resistance.

Oxygen-dependent regulation of tumor growth and metastasis in human breast cancer xenografts.

BACKGROUND: Tumor hypoxia is relevant for tumor growth, metabolism, resistance to chemotherapy and metastasis. We have previously shown that hyperoxia, using hyperbaric oxygen treatment (HBOT), attenuates tumor growth and shifts the phenotype from mesenchymal to epithelial (MET) in the DMBA-induced mammary tumor model. This study describes the effect of HBOT on tumor growth, angiogenesis, chemotherapy efficacy and metastasis in a triple negative MDA-MB-231 breast cancer model, and evaluates tumor growth using a triple positive BT-474 breast cancer model. MATERIALS AND METHODS: 5 x 105 cancer cells were injected s.c. in the groin area of NOD/SCID female mice. The BT-474 group was supplied with Progesterone and Estradiol pellets 2-days prior to tumor cell injection. Mice were divided into controls (1 bar, pO2 = 0.2 bar) or HBOT (2.5 bar, pO2 = 2.5 bar, 90 min, every third day until termination of the experiments). Treatment effects were determined by assessment of tumor growth, proliferation (Ki67-staining), angiogenesis (CD31-staining), metastasis (immunostaining), EMT markers (western blot), stromal components collagen type I, Itgb1 and FSP1 (immunostaining) and chemotherapeutic efficacy (5FU). RESULTS: HBOT significantly suppressed tumor growth in both the triple positive and negative tumors, and both MDA-MB-231 and BT-474 showed a decrease in proliferation after HBOT. No differences were found in angiogenesis or 5FU efficacy between HBOT and controls. Nevertheless, HBOT significantly reduced both numbers and total area of the metastastatic lesions, as well as reduced expression of N-cadherin, Axl and collagen type I measured in the MDA-MB-231 model. No change in stromal Itgb1 and FSP1 was found in either tumor model. CONCLUSION: Despite the fact that behavior and prognosis of the triple positive and negative subtypes of cancer are different, the HBOT had a similar suppressive effect on tumor growth, indicating that they share a common oxygen dependent anti-tumor mechanism. Furthermore, HBOT significantly reduced the number and area of metastatic lesions in the triple negative model as well as a significant reduction in the EMT markers N-cadherin, Axl and density of collagen type I.

Adaptive mechanisms of resistance to anti-neoplastic agents.

Intrinsic and acquired resistance to conventional and targeted therapeutics is a fundamental reason for treatment failure in many cancer patients. Targeted approaches to overcome chemoresistance as well as resistance to targeted approaches require in depth understanding of the underlying molecular mechanisms. The anti-cancer activity of a drug can be limited by a broad variety of molecular events at different levels of drug action in a cell-autonomous and non-cell-autonomous manner. This review summarizes recent insights into the adaptive mechanisms used by tumours to resist therapy including cellular phenotypic plasticity, dynamic alterations of the tumour microenvironment, activation of redundant signal transduction pathways, modulation of drug target expression levels, and exploitation of pro-survival responses.