Mathematical modelling of the use of macrophages as vehicles for drug delivery to hypoxic tumour sites

Poor drug delivery and low rates of cell proliferation are two factors associated with hypoxia that diminish the efficacy of many chemotherapeutic drugs. Since macrophages are known to migrate specifically towards, and localize within, hypoxic tumour regions, a promising resolution to these problems involves genetically engineering macrophages to perform such anti-tumour functions as inducing cell lysis and inhibiting angiogenesis. In this paper we outline a modelling approach to characterize macrophage infiltration into early avascular solid tumours, and extensions to study the interaction of these cells with macrophages already present within the tumour. We investigate the role of chemotaxis and chemokine production, and the efficacy of macrophages as vehicles for drug delivery to hypoxic tumour sites. The model is based upon a growing avascular tumour spheroid, in which volume is filled by tumour cells, macrophages and extracellular material, and tumour cell proliferation and death is regulated by nutrient diffusion. Crucially, macrophages occupy volume, and hence contribute to the volume balance and hence the size of the tumour. We also include oxygen-dependent production of macrophage chemokines, which can lead to accumulations in the hypoxic region of the tumour. We find that the macrophage chemotactic sensitivity is a key determinant of macrophage infiltration and tumour size. Although increased infiltration should be beneficial from the point of view of macrophage-based therapies, such infiltration in fact leads to increased tumour sizes. Finally, we include terms representing the induced death of tumour cells by hypoxic engineered macrophages. We demonstrate that reductions in tumour size can be achieved, but predict that a combination of therapies would be required for complete eradication. We also highlight some counter-intuitive predictions-for example, absolute and relative measures of tumour burden lead to different conclusions about prognosis. In summary, this paper illustrates how mathematical models may be used to investigate promising macrophage-based therapies.     

The Evolution of Tumour Composition During Fractionated Radiotherapy: Implications for Outcome

Current protocols for delivering radiotherapy are based primarily on tumour stage and nodal and metastases status, even though it is well known that tumours and their microenvironments are highly heterogeneous. It is well established that the local oxygen tension plays an important role in radiation-induced cell death, with hypoxic tumour regions responding poorly to irradiation. Therefore, to improve radiation response, it is important to understand more fully the spatiotemporal distribution of oxygen within a growing tumour before and during fractionated radiation. To this end, we have extended a spatially resolved mathematical model of tumour growth, first proposed by Greenspan (Stud Appl Math 51:317–340, 1972), to investigate the effects of oxygen heterogeneity on radiation-induced cell death. In more detail, cell death due to radiation at each location in the tumour, as determined by the well-known linear-quadratic model, is assumed also to depend on the local oxygen concentration. The oxygen concentration is governed by a reaction-diffusion equation that is coupled to an integro-differential equation that determines the size of the assumed spherically symmetric tumour. We combine numerical and analytical techniques to investigate radiation response of tumours with different intratumoral oxygen distribution profiles. Model simulations reveal a rapid transient increase in hypoxia upon regrowth of the tumour spheroid post-irradiation. We investigate the response to different radiation fractionation schedules and identify a tumour-specific relationship between inter-fraction time and dose per fraction to achieve cure. The rich dynamics exhibited by the model suggest that spatial heterogeneity may be important for predicting tumour response to radiotherapy for clinical applications.     

HAX1 maintains the glioma progression in hypoxia through promoting mitochondrial fission

HCLS1-associated protein X-1 (HAX1), an anti-apoptotic molecular, overexpresses in glioma. However, the role of HAX1 in glioma cell surviving in hypoxic environment remains unclear. Western blotting, qRT-PCR, Transwell assay, TUNEL assay, wounding healing assay, clone formation, tumour xenograft model and immunohistochemical staining were used to investigate the role of HAX1 in glioma. HAX1 regulated by HIF-1α was increased in glioma cells cultured in hypoxia. Silencing of HAX1 could cause an increased apoptosis of glioma cells cultured in hypoxia. Silencing of HAX1 also decreased the proliferation, migration and invasion of glioma cells cultured in hypoxia. Increased mitochondrial fission could prevent glioma cells from the damage induced by HAX1 knockdown in hypoxia. Furthermore, HAX1 was found to regulate glioma cells through phosphorylated AKT/Drp signal pathway. In conclusion, our study suggested that HAX1 promoted survival of glioma cells in hypoxic environment via AKT/Drp signal pathway. Our study also provided a potential therapeutic target for glioma.     

Effects of mitomycin C and porfiromycin on exponentially growing and plateau phase cultures

Abstract. Laboratory studies and clinical trials are exploring the use of hypoxia-directed cytotoxic agents as adjuncts to radiotherapy. Because hypoxia and the microenviron-mental inadequacies associated with hypoxia in solid tumours inhibit cell proliferation, an essential requirement for the successful use of hypoxia-directed drugs in cancer therapy is that these drugs be toxic to quiescent tumour cells, as well as tumour cells progressing rapidly through the cell cycle. The experiments reported here compared the cytotoxicities of mitomycin C and porfiromycin to exponentially growing and plateau phase cultures of EMT6 mouse mammary tumour cells. The proliferative status of the cultures did not influence the cytotoxicity of mitomycin C under either aerobic or hypoxic conditions, or the cytotoxicity of porfiromycin in air. Exponentially growing cultures were slightly more sensitive than plateau phase cultures to porfiromycin in hypoxia, but the difference between the sensitivities of proliferating and quiescent cells was much smaller than the difference between aerobic and hypoxic cells. No evidence for repair of potentially lethal damage was found after treatment with porfiromycin in air or in hypoxia; this is in agreement with previous findings for mitomycin C. Mitomycin C and porfiromycin therefore exhibit the toxicity to quiescent cells needed for effective use as hypoxia-directed drugs for the treatment of solid tumours.     

Decursin promotes HIF-1α proteasomal degradation and immune responses in hypoxic tumour microenvironment

BackgroundHypoxia and HIF-1α are important regulators of tumour growth and angiogenesis and could be attractive targets for cancer therapeutics. Decursin is an active compound extracted from the roots of Angelica gigas and has been shown to have potent anti-cancer and anti-angiogenic activities. However, whether decursin regulates HIF-1α activity and immune responses under hypoxic conditions is not yet understood.PurposeThe aim of this study was to identify whether decursin exhibits anti-cancer activity by targeting HIF-1α.Study designWe investigated whether decursin regulates HIF-1α protein stability and increases its degradation. In addition, we determined if decursin increases immune responses in tumour microenvironment to identify its hypoxia-associated anti-cancer activities.Materials and methodsWe performed the hypoxia-responsive element promoter–reporter assay, Western blot analysis, immune-fluorescence assay, semi-quantitative RT-PCR and ELISA for VEGF secretion, CCK-8 assay for cell proliferation, TUNEL assay for apoptosis and invasion assay in A549 human lung cancer or HCT116 human colon cancer cells. In vivo Lewis lung carcinoma (LLC) allograft mouse model was used to check tumour growth and immune responses in tumour microenvironment by immunohistochemistry analysis.ResultsWe observed that decursin inhibited HIF-1 activation under hypoxia by down-regulating the protein level of its subunit HIF-1α. It increased oxygen-dependant hydroxylation and ubiquitination of HIF-1α to promote HIF-1α degradation. Decursin also decreased mRNA expression of HIF-1α target genes. Decursin suppressed cancer cell proliferation, induced apoptosis and inhibited cancer cell invasion under hypoxia in cancer cells. In the allograft mouse tumour model, decursin reduced the hypoxic area and HIF-1α and PD-L1 expression. Infiltrating T cells (CD3+), helper T cells (CD4+) and cytotoxic (CD8+) T cells were accumulated, but regulatory T cells (Foxp3) and myeloid-derived suppressor cell-mediated immune suppressors (Arg1) were attenuated by decursin.ConclusionOur results suggest that decursin is a novel HIF-1α inhibitor that functions by promoting its proteasomal degradation and that it also helps improve T cell activation in tumour microenvironment; these findings provide new explanations about its anti-cancer and anti-angiogenic activity mechanisms.     

Mathematical modelling of drug transport in tumour multicell spheroids and monolayer cultures

In this paper we adapt an avascular tumour growth model to compare the effects of drug application on multicell spheroids and on monolayer cultures. The model for the tumour is based on nutrient driven growth of a continuum of live cells, whose birth and death generates volume changes described by a velocity field. The drug is modelled as an externally applied, diffusible material capable of killing cells, both linear and Michaelis-Menten kinetics for drug action on cells being studied. Numerical solutions of the resulting system of partial differential equations for the multicell spheroid case are compared with closed form solutions of the monolayer case, particularly with respect to the effects on the cell kill of the drug dosage and of the duration of its application. The results show an enhanced survival rate in multicell spheroids compared to monolayer cultures, consistent with experimental observations, and indicate that the key factor determining this is drug penetration. An analysis of the large time tumour spheroid response to a continuously applied drug at fixed concentration reveals up to three stable large time solutions, namely the trivial solution (i.e. a dead tumour), a travelling wave (continuously growing tumour) and a sublinear growth case in which cells reach a pseudo-steady-state in the core. Each of these possibilities is formulated and studied, with the bifurcations between them being discussed. Numerical solutions reveal that the pseudo-steady-state solutions persist to a significantly higher drug dose than travelling wave solutions.     

Pronounced hypoxia in models of murine and human leukemia: high efficacy of hypoxia-activated prodrug PR-104

Recent studies indicate that interactions between leukemia cells and the bone marrow (BM) microenvironment promote leukemia cell survival and confer resistance to anti-leukemic drugs. There is evidence that BM microenvironment contains hypoxic areas that confer survival advantage to hematopoietic cells. In the present study we investigated whether hypoxia in leukemic BM contributes to the protective role of the BM microenvironment. We observed a marked expansion of hypoxic BM areas in immunodeficient mice engrafted with acute lymphoblastic leukemia (ALL) cells. Consistent with this finding, we found that hypoxia promotes chemoresistance in various ALL derived cell lines. These findings suggest to employ hypoxia-activated prodrugs to eliminate leukemia cells within hypoxic niches. Using several xenograft models, we demonstrated that administration of the hypoxia-activated dinitrobenzamide mustard, PR-104 prolonged survival and decreased leukemia burden of immune-deficient mice injected with primary acute lymphoblastic leukemia cells. Together, these findings strongly suggest that targeting hypoxia in leukemic BM is feasible and may significantly improve leukemia therapy.     

Cell migration in multicell spheroids: Swimming against the tide

Multicell spheroids, small spherical clusters of cancer cells, have become an importantin vitro model for studying tumour development given the diffusion limited geometry associated with many solid tumour growths. Spheroids expand until they reach a dormant state where they exhibit a grossly static three-layered structure. However, at a cellular level, the spheroid is demonstrably dynamic with constituent cells migrating from the outer well-nourished region of the spheroid toward the necrotic central core. The mechanism that drives the migrating cells in the spheroid is not well understood. In this paper we demonstrate that recent experiments on internationalization can be adequately described by implicating pressure gradients caused by differential cell proliferation and cell death as the primary mechanism. Although chemotaxis plays a role in cell movement, we argue that it acts against the passive movement caused by pressure differences.     

miR-210 is induced by hypoxia and regulates neural cell adhesion molecule in prostate cells

Hypoxia in prostate tumours has been associated with disease progression and metastasis. MicroRNAs are short noncoding RNA molecules that are important in several cell processes, but their role in hypoxic signalling is still poorly understood. miR-210 has been linked with hypoxic mechanisms, but this relationship has been poorly characterised in prostate cancer. In this report, the link between hypoxia and miR-210 in prostate cancer cells is investigated. Polymerase chain reaction analysis demonstrates that miR-210 is induced by hypoxia in prostate cancer cells using in vitro cell models and an in vivo prostate tumour xenograft model. Analysis of The Cancer Genome Atlas prostate biopsy datasets shows that miR-210 is significantly correlated with Gleason grade and other clinical markers of prostate cancer progression. Neural cell adhesion molecule (NCAM) is identified as a target of miR-210, providing a biological mechanism whereby hypoxia-induced miR-210 expression can contribute to prostate cancer. This study provides evidence that miR-210 is an important regulator of cell response to hypoxic stress and proposes that its regulation of NCAM may play an important role in the pathogenesis of prostate cancer.     

The Importance of GLUT3 for De Novo Lipogenesis in Hypoxia-Induced Lipid Loading of Human Macrophages

Atherosclerotic lesions are characterized by lipid-loaded macrophages (foam cells) and hypoxic regions. Although it is well established that foam cells are produced by uptake of cholesterol from oxidized LDL, we previously showed that hypoxia also promotes foam cell formation even in the absence of exogenous lipids. The hypoxia-induced lipid accumulation results from increased triglyceride biosynthesis but the exact mechanism is unknown. Our aim was to investigate the importance of glucose in promoting hypoxia-induced de novo lipid synthesis in human macrophages. In the absence of exogenous lipids, extracellular glucose promoted the accumulation of Oil Red O-stained lipid droplets in human monocyte-derived macrophages in a concentration-dependent manner. Lipid droplet accumulation was higher in macrophages exposed to hypoxia at all assessed concentrations of glucose. Importantly, triglyceride synthesis from glucose was increased in hypoxic macrophages. GLUT3 was highly expressed in macrophage-rich and hypoxic regions of human carotid atherosclerotic plaques and in macrophages isolated from these plaques. In human monocyte-derived macrophages, hypoxia increased expression of both GLUT3 mRNA and protein, and knockdown of GLUT3 with siRNA significantly reduced both glucose uptake and lipid droplet accumulation. In conclusion, we have shown that hypoxia-induced increases in glucose uptake through GLUT3 are important for lipid synthesis in macrophages, and may contribute to foam cell formation in hypoxic regions of atherosclerotic lesions.     

PD-1 and TIGIT blockade differentially affect tumour cell survival under hypoxia and glucose deprived conditions in oesophageal adenocarcinoma; implications for overcoming resistance to PD-1 blockade in hypoxic tumours

Recent studies have demontrated that immune checkpoint receptors are expressed on the surface of oesophageal adenocarcinoma (OAC) cells and might confer a survival advantage. This study explores the role of PD-1 and TIGIT signalling in OAC cells in either promoting or inhibiting the survival of OAC cells under characteristic features of the tumour microenvironment including nutrient-deprivation and hypoxia. PD-1 and TIGIT are expressed in normal and pre-malignant oesophageal epithelial cells and this expression significantly decreases along the normal- Barrett's Oesophagus- OAC disease sequence. However, glucose-deprivation and hypoxia significantly upregulated PD-1 and TIGIT on the surface of OAC cells in vitro. PD-1 blockade decreased OAC cell proliferation under normoxia but enhanced proliferation and decreased cell death in OAC cells under hypoxia and glucose-deprivation. TIGIT blockade decreased proliferation and induced OAC cell death, an effect that was maintained under nutrient-deprivation and hypoxia. Basal respiration and glycolytic reserve were enhanced and GLUT1 was upregulated on the surface of a subpopulation of OAC cells following PD-1 blockade. In contrast, TIGIT blockade enhanced a glycolytic phenotype in OAC cells, yet decreased other metabolic parameters including oxidative phosphorylation and basal respiration. Interestingly, inhibition of oxidative phosphorylation significantly upregulated TIGIT expression and inhibition of oxidative phosphorylation and glycolysis significantly decreased PD-1 on the surface of a subpopulation of OAC cells in vitro. These findings suggest an immune-independent mechanism for PD-1 inhibitor resistance in hypoxic tumours and suggest that TIGIT might be a more effective therapeutic target in OAC compared with PD-1 for treating hypoxic tumours.     

Hypoxia and reoxygenation: A pressure for mutant p53 cell selection and tumour progression

Recent findings indicate that in a hypoxic environment, oncogenically transformed cells with a mutant form of the tumour suppressor gene p53 may have a survival advantage over similar cells with wild-type p53. This is because the extent of hypoxia-induced apoptosis has been observed to diminish with the loss of wild-type p53 function in certain cell lines. Hypoxic conditions, common in most solid tumours, may thus provide a physiological pressure to select for cells with mutations in the p53 gene. A new model incorporating cell-specific parameters is proposed here to quantify the survival advantage of mutant or null p53 cells over their wild-type counterparts at any level of oxygen deprivation. Predictions are in good agreement with previous monolayer culture experiments comparing hypoxic survival of null and wild-type p53 cells. By extending the model we are able to investigate the effects of repeated rounds of hypoxia and reoxygenation on a mixture of wild-type and mutant or null p53 cells and determine how many rounds are required before a subpopulation of mutant or null p53 cells overtakes a given population of wild-type p53 cells.     

Hypoxia Enhances the Proliferative Response of Macrophages to CSF-1 and Their Pro-Survival Response to TNF

In chronic inflammatory lesions there are increased numbers of macrophages with a possible contribution of enhanced survival/proliferation due, for example, to cytokine action; such lesions are often hypoxic. Prior studies have found that culture in low oxygen can promote monocyte/macrophage survival. We show here, using pharmacologic inhibitors, that the hypoxia-induced pro-survival response of macrophages exhibits a dependence on PI3-kinase and mTOR activities but surprisingly is suppressed by Akt and p38 MAPK activities. It was also found that in hypoxia at CSF-1 concentrations, which under normoxic conditions are suboptimal for macrophage proliferation, macrophages can proliferate more strongly with no evidence for alteration in CSF-1 receptor degradation kinetics. TNF promoted macrophage survival in normoxic conditions with an additive effect in hypoxia. The enhanced hypoxia-dependent survival and/or proliferation of macrophages in the presence of CSF-1 or TNF may contribute to their elevated numbers at a site of chronic inflammation.     

Inhibition of sphingosine kinase 1 suppresses proliferation of glioma cells under hypoxia by attenuating activity of extracellular signal-regulated kinase

Objectives: Sphingosine kinase (SphK), which is regulated by hypoxia, catalyses phosphorylation of sphingosine to produce sphingosine‐1‐phosphate, which stimulates invasiveness of gliomas. However, whether SphK is involved in proliferation of glioma cells under hypoxic conditions is not clearly understood. In this study, we have investigated the role of SphK in of proliferation glioma cells under hypoxia. Materials and methods: Effects of small interfering RNA (siRNA) on SphKs, SKI (inhibitor of SphK) and U0126 (inhibitor of ERK) on proliferation of glioma cells under hypoxia were studied using CCK‐8 assay and flow cytometry. Protein expression profiles were evaluated by Western blot analysis. Results:  SKI suppressed proliferation of glioma cells under hypoxia. Similarly, downregulation of SphKs by siRNA inhibited glioma cell proliferation, and the cell cycle was arrested in G₂/M phase when SphK1 was inhibited. In addition, inhibition of SphK1 attenuated phosphorylation of ERK in hypoxic conditions. Furthermore, U0126 markedly inhibited cell population growth and arrested cells in G₂/M as effectively as SKI. However, silencing SphK2 induced cell cycle arrest in the S phase and it showed little effect on hypoxia‐induced activation of ERK. Conclusions: SphK1 and SphK2 are involved in proliferation of glioma cells in hypoxic conditions through distinct signalling pathways. SphK1, but not SphK2, promotes cell population expansion in hypoxic conditions by activating ERK.     

MicroRNA-1 facilitates hypoxia-induced injury by targeting NOTCH3

Cell proliferation, apoptosis, and autophagy have been reported to be related to myocardial ischemia injury. MicroRNAs have attracted wide attention on regulating cell proliferation, apoptosis, and autophagy. miR-1 expression has been reported to be dysregulated in cardiac tissue or cells with hypoxia, while the exact roles as well as underlying mechanism remain poorly understood. In this study, we investigated the potential roles of miR-1 in cell proliferation, apoptosis, and autophagy in hypoxia-treated cardiac injury and explored the underlying mechanism using H9c2 cells. Results showed that hypoxic stimulation inhibited cell proliferation and the expression of miR-1 but promoted cell apoptosis in H9c2 cells. Moreover, overexpression of miR-1 promoted cell apoptosis and inhibited cell proliferation and autophagy in H9c2 cells treated with hypoxia, while its knockdown played an opposite effect. In addition, bioinformatics, luciferase reporter, and RNA immunoprecipitation analyses indicated that NOTCH3 was a direct target of miR-1 and its upregulation reversed the effects of miR-1 on cell proliferation, apoptosis, and autophagy in hypoxia-treated H9c2 cells. Taken together, our data suggested that miR-1 promoted hypoxia-induced injury by targeting NOTCH3, indicating novel therapeutic targets for treatment of myocardial ischemia injury.     

CAIX furthers tumour progression in the hypoxic tumour microenvironment of esophageal carcinoma and is a possible therapeutic target

The hypoxic tumour microenvironment of solid tumours represents an important starting point for modulating progression and metastatic spread. Carbonic anhydrase IX (CAIX) is a known HIF-1α-dependent key player in maintaining cell pH conditions under hypoxia. We show that CAIX is strongly expressed in esophageal carcinoma tissues. We hypothesize that a moderate CAIX expression facilitates metastases and thereby worsens prognosis. Selective inhibition of CAIX by specific CAIX inhibitors and a CAIX knockdown effectively inhibit proliferation and migration in vitro. In the orthotopic esophageal carcinoma model, the humanized HER2 antibody trastuzumab down-regulates CAIX, possibly through CAIX’s linkage with HER2 in the hypoxic microenvironment. Our results show CAIX to be an essential part of the tumour microenvironment and a possible master regulator of tumour progression. This makes CAIX a highly effective and feasible therapeutic target for selective cancer treatment.     

Hypoxia sensitizes human malignant glioma cells towards CD95L-induced cell death

Death ligands such as CD95 ligand (CD95L) have limited activity against glioma cells under normoxic conditions. Hypoxia is a critical aspect of the microenvironment of gliomas in vivo. We investigated the effect of co-exposure to acute hypoxia and CD95 ligand in three human malignant glioma cell lines with different susceptibility to CD95L under normoxic conditions. Hypoxia sensitized all three cell lines towards CD95L-induced cell death. Co-exposure resulted in apoptotic changes in the early phase, with gradual conversion to secondary necrosis with increasing length of hypoxia. The mitochondrial injury induced by hypoxia was enhanced by co-treatment, and caspase cleavage became prominent. Inhibition of the epidermal growth factor receptor (EGFR), although sensitizing glioma cells to CD95L under normoxia, protects glioma cells from hypoxia by reducing energy consumption. However, the opposing effects of EGFR signalling on death induced by CD95L or hypoxia were neutralized by co-exposure to hypoxia and CD95L. Furthermore, inhibition of protein synthesis by cycloheximide also reduced glucose consumption and conferred protection from hypoxia, but did not modulate CD95L-induced cell death under hypoxic conditions. These results suggest that death ligands may be useful to target hypoxic tumour cells resistant to conventional therapies or to complement strategies aiming at the induction of tumour hypoxia.     

piRNA‐63076 contributes to pulmonary arterial smooth muscle cell proliferation through acyl‐CoA dehydrogenase

Piwi‐interacting RNAs (piRNAs) are thought to be germline‐specific and to be involved in maintaining genome stability during development. Recently, piRNA expression has been identified in somatic cells in diverse organisms. However, the roles of piRNAs in pulmonary arterial smooth muscle cell (PASMC) proliferation and the molecular mechanism underlying the hypoxia‐regulated pathological process of pulmonary hypertension are not well understood. Using hypoxic animal models, cell and molecular biology, we obtained the first evidence that the expression of piRNA‐63076 was up‐regulated in hypoxia and was positively correlated with cell proliferation. Subsequently, we showed that acyl‐CoA dehydrogenase (Acadm), which is negatively regulated by piRNA‐63076 and interacts with Piwi proteins, was involved in hypoxic PASMC proliferation. Finally, Acadm inhibition under hypoxia was partly attributed to DNA methylation of the Acadm promoter region mediated by piRNA‐63076. Overall, these findings represent invaluable resources for better understanding the role of epigenetics in pulmonary hypertension associated with piRNAs.