Tumor Microregions Research Articles

Use of atomic force microscopy to assess the biomechanical properties of 3D tumor cell models

Multicellular spheroids are a unique object-model for toxicological studies. Cells in a 3D cluster have microenvironment, intercellular communication, which allows spheroids to be used as more realistic model compared to traditional cell cultures. It is known that tumor microregions consist of heterogeneous populations of cancer cells, in which cell growth and response to antitumor drugs depend on their 3D architecture, intercellular contacts and interaction with the microenvironment. In addition, tumor growth and progression are also strongly influenced by mechanical cues Currently, 3D cell culture models are a powerful tool for studying the toxicity of drug compounds and nanomaterials of different composition and morphology. This review presents data on the use of various techniques, in particular atomic force microscopy, to investigate changes in the mechanical properties of cells in spheroids. In particular, the use of atomic force microscopy as a tool to reveal physicochemical parameters of cells during pathophysiological processes or drug exposure is considered. The relevance of this review is related to the increasing interest in the role of biomechanical properties of tissues, cells and subcellular structures as markers of pathophysiological conditions.

Temperature and Tumor Microenvironment Dual Responsive Mesoporous Magnetic Nanospheres for Magnetothermal Effect-Induced Cancer Theranostics

Temperature and Tumor Microenvironment Dual Responsive Mesoporous Magnetic Nanospheres for Magnetothermal Effect-Induced Cancer Theranostics

For the Success of Oncolytic Viruses: Single Cycle Cures or Repeat Treatments? (One Cycle Should Be Enough).

For the Success of Oncolytic Viruses: Single Cycle Cures or Repeat Treatments? (One Cycle Should Be Enough).

Spatio-Temporal Dynamics of Hypoxia during Radiotherapy

Tumour hypoxia plays a pivotal role in cancer therapy for most therapeutic approaches from radiotherapy to immunotherapy. The detailed and accurate knowledge of the oxygen distribution in a tumour is necessary in order to determine the right treatment strategy. Still, due to the limited spatial and temporal resolution of imaging methods as well as lacking fundamental understanding of internal oxygenation dynamics in tumours, the precise oxygen distribution map is rarely available for treatment planing. We employ an agent-based in silico tumour spheroid model in order to study the complex, localized and fast oxygen dynamics in tumour micro-regions which are induced by radiotherapy. A lattice-free, 3D, agent-based approach for cell representation is coupled with a high-resolution diffusion solver that includes a tissue density-dependent diffusion coefficient. This allows us to assess the space- and time-resolved reoxygenation response of a small subvolume of tumour tissue in response to radiotherapy. In response to irradiation the tumour nodule exhibits characteristic reoxygenation and re-depletion dynamics which we resolve with high spatio-temporal resolution. The reoxygenation follows specific timings, which should be respected in treatment in order to maximise the use of the oxygen enhancement effects. Oxygen dynamics within the tumour create windows of opportunity for the use of adjuvant chemotherapeutica and hypoxia-activated drugs. Overall, we show that by using modelling it is possible to follow the oxygenation dynamics beyond common resolution limits and predict beneficial strategies for therapy and in vitro verification. Models of cell cycle and oxygen dynamics in tumours should in the future be combined with imaging techniques, to allow for a systematic experimental study of possible improved schedules and to ultimately extend the reach of oxygenation monitoring available in clinical treatment.

Abstract A108: Induction of mitochondrial dysfunction as a strategy for targeting tumor cells in hypoxic microenvironment.

Abstract A fundamental problem in cancer drug discovery is to identify compounds that eliminate dormant malignant cells responsible for tumor relapse. Abnormal vascularization of solid tumors leads to the generation of tissue microenvironments that are chronically starved of oxygen and nutrients. Cells residing in such environments are slowly growing or quiescent and display altered phenotypic characteristics when compared to cells located in more vascularized regions. Such non-dividing cells are often resistant to mainstay standard chemotherapies that rely on DNA replication and cell division to elicit their anti-tumor effect. The altered phenotype of quiescent cells enables them to survive chemotherapeutic regimes and reseed nascent tumors following secession of chemotherapy. Indeed resistance to agents such as doxorubicin, cisplatin and vinblastine has been correlated with poor vasculature, tumor relapse and poor patient survival. Thus there is a need to alter the scope of cancer drug discovery and focus more on screening for agents that can exploit the altered phenotype of quiescent metabolically stressed cells to eliminate tumors. Cell-based screening for novel anticancer drugs is typically performed using monolayer cultures of tumor cells, however such monolayer cultures do not represent the characteristics of 3-D solid tumors, frequently leading to the failure of subsequent in vivo models. The multicellular tumor spheroid model is of intermediate complexity between in vivo tumors and in vitro monolayer cultures and is more suitable for drug screening and evaluation. Spheroids are known to be more resistant to drug effects compared to monolayer cultures. Resistance is due not only to pharmacokinetic obstacles limiting drug penetrance to inner layers, but also to multi-cellular interactions leading to altered expression of genes and proteins regulating drug response. An additional benefit of growing cells three- dimensionally is the opportunity to explore the core regions’ potential vulnerability related to hypoxia and nutrient defiency and to reflect the heterogeneous milieu in tumor microregions relative to the supply. Poorly vascularized and perfused tumor microareas in many aggressive cancers have limited access not only to oxygen but also to glucose. Core regions are also associated with acidic pHsince these tumor cells change their metabolism towards increased glycolysis, resulting in increased lactic acid production. We here employed spheroid cultures of HCT116 colon cancer cells to screen a diverse chemical library with the aim to find compounds with cytotoxic activity in core, hypoxic, regions. The screen identified a compound (VLX600) demonstrating anti-cancer activity with a large therapeutic window both in vitro and in vivo. Here we characterized the mechanism of action and potential of VLX600 as anti-cancer therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A108. Citation Format: Xiaonan Zhang, Mårten Fryknäs, Emma Hernlund, Walid Fayad, Angelo De Milito, Maria Hägg Olofsson, Stig Linder. Induction of mitochondrial dysfunction as a strategy for targeting tumor cells in hypoxic microenvironment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A108.

Tissue Distribution and Efficacy of Gold Nanorods Coupled with Laser Induced Photoplasmonic Therapy in Ehrlich Carcinoma Solid Tumor Model

Gold nanorods (GNR) within tumor microregions are characterized by their ability to absorb near IR light and emit heat in what is called photoplasmonic effect. Yet, the efficacy of nanoparticles is limited due to intratumoral tissue distribution reasons. In addition, distribution of GNRs to normal tissue might result in non specific toxicity. In the current study, we are assessing the intratumoral and tissue distribution of PEGylated GNRs on the top of its antitumor characteristics when given intravenously or intratumoral to solid tumor bearing mice and coupled with laser photoplasmonic sessions. PEGylated GNRs with a longitudinal size of less than 100 nm were prepared with aspect ratio of 4.6 showing strong surface plasmon absorption at wavelength 800 nm. Pharmacokinetics of GNR after single I.V. administration (0.1 mg/kg) showed very short systemic circulating time (less than 3 h). On the other hand, tissue distribution of I.V. GNR (0.1 mg/kg) to normal animals showed preferential deposition in spleen tissue. Repeated administration of I.V. GNR resulted in preferential accumulation in both liver and spleen tissues. In addition, I.V. administration of GNR to Ehrlich carcinoma tumor bearing mice resulted in similar tissue distribution; tumor accumulation and anti-tumor effect compared to intratumoral administration. In conclusion, the concentration of GNR achieved within tumors microregions after I.V. administration was comparable to I.T. administration and sufficient to elicit tumoral growth arrest when coupled with laser-aided photoplasmonic treatment.

Idarubicin and Idarubicinol Effects on Breast Cancer Multicellular Spheroids

Despite extensive preclinical evaluation in several experimental models, no studies have determined the effect of idarubicin and its metabolite idarubicinol on multicellular spheroids, a model which mimics the microregions of solid tumors. The principal aim of the present study was to investigate the in vitro cytotoxicity of idarubicin and its metabolite idarubicinol on MCF-7 breast cancer cells growing as monolayers or multicellular spheroids and to evaluate the influence of the length of exposure on the cytotoxic effect of both drugs. Cytoxicity was evaluated on monolayer and spheroid cultures exposed to idarubicin and idarubicinol 0.01-1000 ng/ml for 24 h or treated for 6, 12, 24 and 48 h to 100 ng/ml of both drugs. The IC50 of idarubicin and idarubicinol were 3.3±0.4 and 3.6±0.7 ng/ml, respectively, on MCF-7 monolayers and 7.9±1.1 and 5.3±0.7 ng/ml in multicellular spheroids, respectively. The antiproliferative effects of 100 ng/ml idarubicin and idarubicinol on MCF-7 spheroids was characterized by a marked time-dependence, which was less evident on MCF-7 growing as monolayer. In conclusion, the present experimental data demonstrate, for the first time, that idarubicin and idarubicinol have significant cytotoxic activity against multicellular spheroids, comparable to the antiproliferative effects on monolayer cells. In contrast, spheroids displayed substantial resistance after short exposure times that was not present in the two dimensional cultures.

A model of quiescent tumour microregions for evaluating multicellular resistance to chemotherapeutic drugs

The quiescent cell population of tumours poses a barrier to the success of many cancer therapies. Most chemotherapeutic drugs target proliferating cells, but the growth fraction of many tumours is low. Based on the multicellular tumour spheroid model, a system was developed using human colon adenocarcinoma (DLD-1) cells to mimic the microenvironment of quiescent microregions of solid tumours. The quiescent tumour spheroids (TSQ) showed decreased expression of the proliferation marker Ki-67 and increased expression of the quiescence marker p27kip1 compared to proliferating spheroids (TSP). The quiescent status of the TSQ was confirmed by long-term growth assessment. The quiescence was completely reversible demonstrating that the TSQ retained the ability to proliferate and morphological assessment by light microscopy confirmed the absence of significant apoptosis. When the efficacy of widely used chemotherapeutic drugs was determined, vinblastine, doxorubicin, cisplatin and 5-fluorouracil (5-FU) all produced significant cell death in the TSP. However, while still effective, the potencies of doxorubicin and cisplatin were significantly reduced in TSQ. In contrast, 5-FU and vinblastine did not produce cell death in the TSQ. In summary, TSQ show considerable resistance to a panel of established chemotherapeutic agents and represent a useful model for evaluating the efficacy of drugs and other cancer therapies in quiescent tumours.

A Single-Cell Approach in Modeling the Dynamics of Tumor Microregions

Interactions between tumor cells and their environment lead to the formation of microregions containing nonhomogeneous subpopulations of cells and steep gradients in oxygen, glucose, and other metabolites. To address the formation of tumor microregions on the level of single cells, I propose a new two-dimensional time-dependent mathematical model taking explicitly into account the individually regulated biomechanical processes of tumor cells and the effect of oxygen consumption on their metabolism. Numerical simulations of the self-organized formation of tumor microregions are presented and the dynamics of such a process is discussed.

Differential effects of buthionine sulphoximine in hypoxic and non-hypoxic regions of human cervical carcinoma xenografts

Background and Purpose: Recently we reported increased glutathione (GSH) levels in hypoxic regions of ME 180 and SiHa cervical cancer xenografts. Since this association might act synergistically to protect from radiotherapy, we examined the differential effects of the GSH depleting agent buthionine suiphoximine (BSO) in relation to tumor oxygenation. Materials and methods: The nitroimidazole EF5 was used to label tumor hypoxia. GSH levels were determined in cryostat sections using a sensitive HPLC assay and in parallel sections using fluorescence image analysis. Using a dual-labeling method, GSH levels were determined selectively in hypoxic and non-hypoxic tumor regions. Results: GSH levels were higher in hypoxic than in non-hypoxic regions of cervical carcinoma xenografts. Treatment with BSO produced a more pronounced GSH depletion in regions of hypoxia, resulting in similar post-treatment levels in hypoxic and non-hypoxic areas. Conclusions: BSO effectively depletes GSH in hypoxic microregions of tumors. These findings suggest a potential role for BSO as an adjunct to radiotherapy in cervical cancer patients.

Variable presence of hypoxia in M006 human glioma spheroids and in spheroids and xenografts of clonally derived sublines.

Recently we reported the variable presence of hypoxia adjacent to necrosis in human glioma lines grown as subcutaneous tumours in severe combined immunodeficient (SCID) mice. To assess the basis for this observation, we examined the pattern of oxygenation in M006 and M006XLo glioma spheroids. We found a wide range of binding of [3H]misonidazole to cells adjacent to the necrotic core, analogous to the patterns seen in xenografts, indicating substantial differences in the central oxygen tension of the spheroids. Clonal selection was used to isolate single cell-derived sublines of the M006XLo line. Some sublines gave spheroids that showed narrow distributions of [3H]misonidazole binding to the cells adjacent to necrosis, whereas other sublines showed a range of binding similar to that seen in spheroids of the parent line. After additional passages in monolayer culture, clonal sublines occasionally gave rise to spheroids in which the mean oxygen tension of cells adjacent to necrosis differed substantially from that of the initial spheroids. No relationship was evident between the thickness of the rim of viable cells and the presence or absence of central hypoxia, over a wide range of rim thickness. These results indicate that different oxygenation characteristics of glioma spheroids and tumour microregions are unlikely to arise from stable genetic variants coexisting in the parent line.

The effects of carbogen and nicotinamide on intravascular oxyhaemoglobin saturations in SCCVII and KHT murine tumours.

Considerable effort has been focused on devising methods for manipulating tumour oxygenation and thereby improving tumour radiosensitivity. The combination of nicotinamide and carbogen has been proposed to oxygenate both chronically and acutely hypoxic cells in tumours. However, results have varied markedly with both tumour model and measurement technique. The current objectives were (1) to determine whether changes in radiosensitivity following oxygen manipulation correlated with changes in tumour oxygenation and (2) to assess whether oxygenation was preferentially improved in specific tumour micro-regions. Using two murine tumour lines, the SCCVII carcinoma and the KHT sarcoma, tumour intravascular HbO2 saturations were measured cryospectrophotometrically following nicotinamide, carbogen or the combination. Generally, nicotinamide had minor effects on oxygenation, arguing against a substantial effect on acute hypoxia, while carbogen and the combination produced marked and equivalent improvements in oxygen availability. These results demonstrate that changes in tumour radiosensitivity may not agree with corresponding changes in oxygenation, even within a given tumour model, and that the efficacy of a given manipulative agent may vary substantially with tumour line. One possible explanation for these findings is that different subpopulations of clonogenic vs non-clonogenic cells may be oxygenated by alternative treatments.

PROLIFERATION AND OXYGENATION STATUS OF WIDR SPHEROIDS IN DIFFERENT LACTATE AND OXYGEN ENVIRONMENTS

Human colon adenocarcinoma cells WiDr were cultured as monolayers or multicellular spheroids in 5% or 20% (v/v) oxygen and in various external lactate concentrations of 0-20 mM. Doubling times and H-3-thymidine labeling indices of exponential monolayer cells indicated that there was no difference in growth behavior between the two oxygen environments, yet these parameters reflected a growth retardation upon elevation of lactate. Growth of WiDr spheroids was retarded in both low oxygen and high lactate concentrations. There was a tendency towards a decrease in the thickness of the viable cell rim with increasing lactate in 20% O-2, whereas the width of the viable rim increased significantly as a function of external lactate in 5% O-2. Intraspheroidal oxygen tensions (Po-2) measured with microelectrodes were less in 5% O-2 than in 20% O-2, yet did not vary systematically as a function of external lactate. Po-2 values in the spheroid center dropped to 0 mm Hg prior to the emergence of central necrosis under all conditions investigated. Unlike numerous other spheroid types investigated up to now, WiDr spheroids mimic tumor microregions with hypoxia-induced necrosis and with nonproliferating cells at very low oxygen pressure.

Changes in tumour blood flow, oxygenation and interstitial fluid pressure induced by pentoxifylline.

Pentoxifylline (PTX) has been shown to increase radiation damage to tumours and to decrease late radiation-induced injury to normal tissues. This tumour radiation sensitisation results from increased oxygen supply via improved tumour perfusion. We propose that the improved perfusion results from decreased viscous resistance and/or geometric resistance. The decreased flow resistance may be accompanied by a reduction in microvascular pressure (MVP). Since MVP is approximately equal to the interstitial fluid pressure (IFP), PTX should lead to a decrease in IFP. To test this hypothesis, we measured PO2, laser Doppler flow (RBC flux) and IFP in FSaII murine tumours at two doses (PTX at 25 and 100 mg per kg body weight) which sensitise this tumour to X-irradiation. We found that 25 mg kg-1 PTX was ineffective, but 100 mg kg-1 PTX was effective in increasing the PO2 of this tumour. PTX at 100 mg kg-1 (i.p.) increased median PO2 from 5 to 7 mmHg (P < 0.05) within 2 h, and decreased the fraction of PO2 values < 5 mmHg from 65% to 45% (P < 0.05). In support of our hypothesis, we found that with this dose of PTX, RBC flux in the tumour centre increased significantly (n = 6, P < 0.05) prior to an approximately 40% decrease (n = 13, P < 0.05) in tumour interstitial fluid pressure (TIFP), without changes in mean arterial blood pressure (MABP). In conclusion, a single i.p. administration of PTX at 100 mg kg-1 can increase oxygen availability in the tumour due to ameliorate hypoxia in tumour microregions. Second, PTX can lower the elevated TIFP without lowering the MABP.

Improved tumour oxygenation and radiosensitization by combination with nicotinamide and pentoxifylline.

We hypothesized that the combination of pentoxifylline (PTX) and nicotinamide (NA) can reduce the radioresistance of FSaII murine fibrosarcoma by improving oxygenation of the hypoxic tumour cells. The purpose of this study was to test the hypothesis; first, tumour blood flow after treatment with NA, PTX, and the combination (PTX+NA) was measured using laser Doppler flowmetry. Second, intratumour pO2 after various treatments was measured polarographically using O2 microelectrodes. Third, the radiation response was also measured, i.e. after an exposure to 20 Gy, the time required to reach a four-fold increase in initial tumour volume was 18 days in the saline-treated control group, 21 days in the NA-treated group, 26 days in the PTX-treated group, but was 36 days in PTX+NA treated group. Interestingly, tumour blood flow significantly increased at 10 min after injection of PTX+NA. The mean pO2 in untreated control tumours was 7.5 mmHg, increasing to 13.1 mmHg after 500 mg/kg of NA alone. Repeated injections of PTX with 100 mg/kg/day for 3 days significantly increased intratumour pO2 to 17.2 mmHg. Compared with PTX alone, PTX+NA slightly increased intratumour pO2 from 17.2 to 18.5 mmHg. However, the percentage of regions having values < 2.5 mmHg significantly decreased from 5% with PTX alone to 1% with PTX+NA. In conclusion, single or multiple injections of PTX may increase available O2 in the tumour and thus ameliorate hypoxia in tumour microregions. As previously reported, the subsequent injection of NA may efficiently oxygenate acutely hypoxic cells. Consequently, PTX+NA may increase the radioresponse of tumours by oxygenating chronic as well as acutely hypoxic cells. PTX alone or in combination with NA is potentially useful to radiosensitize tumours.

Biological response of multicellular emt6 spheroids to exogenous lactate

The influence of elevated lactate concentrations, as found in tumor microregions, on cellular growth, viability, and metabolic state was studied employing the multicellular spheroid model. Spheroids of EMT6/Ro cells were cultured at 37 degrees C in 5% or 20% (v/v) oxygen, using stirred media with various concentrations of exogenous lactate ranging from 0.0 mM (standard conditions) to 20.0 mM. Elevated concentrations of exogenous lactate led to a considerable decrease of the maximum spheroid diameter at growth saturation, e.g., for 20% O2 from around 1700 microns to 700 microns in 0.0 and 20.0 mM lactate respectively. Histological investigations showed that the thickness of the viable cell rim was increased by elevated lactate concentrations in 20% O2, whereas this correlation was reversed in 5% O2. Cultivation of spheroids in increasing lactate concentrations was associated with a shift of metabolic pathways from net production to increased utilization of lactate in both 20% and 5% O2, as determined by standard enzymatic assays. Oxygen tension (PO2) values measured with micro-electrodes were less in spheroids cultured in high lactate (9.0 and 20.0 mM) than under standard conditions, irrespective of the external oxygen concentration. This finding reflected a substantial increase in the cellular O2 consumption with elevated external lactate levels. At given lactate concentrations, respiration rates that were derived from measured PO2 distributions by theoretical considerations were significantly lower in 5% O2 than in 20% O2.

Improvement in RBC Flux, Acidosis and Oxygenation in Tumour Microregions by Fluosol-DA 20%

Using laser Doppler flowmetry, we investigated the effects of Fluosol-DA 20% on RBC flux in FSaII tumours and on RBC flux in normal skin of C3H mice. The RBC flux in tumours was significantly increased; however, the RBC flux in normal skin fluctuated only slightly after the treatment with Fluosol-DA 20%. Since an increase in RBC flux by Fluosol-DA 20% may facilitate the removal of acidic metabolites from the tumours, the effects of Fluosol-DA 20% on intratumour pH was also measured. We also measured intratumour pO2 after various dosages of Fluosol-DA 20% with carbogen inhalation. We concluded that the administration of Fluosol-DA 20% selectively increased the RBC flux in tumours compared to that in normal skin. Moreover, carbogen inhalation combined with increasing dosages up to 36 ml/kg of Fluosol-DA 20% effectively enhanced tumour oxygenation in FSaII tumours.

Metabolic imaging in microregions of tumors and normal tissues with bioluminescence and photon counting.

A method has been developed for metabolic imaging on a microscopic level in tumors, tumor spheroids, and normal tissues. The technique makes it possible to determine the spatial distribution of glucose, lactate, and ATP in absolute terms at similar locations within tissues or cell aggregates. The substrate distributions are registered in serial cryostat sections from tissue cryobiopsies or from frozen spheroids with the use of bioluminescence reactions. The light emission is measured directly by a special imaging photon counting system enabling on-line image analysis. The technique has been applied to human breast cancer xenografts, to spheroids originating from a human colon adenocarcinoma, and to skeletal rat muscle. Preliminary data obtained indicate that heterogeneities in the substrate distributions measured are much more pronounced in tumors than in normal tissue. There was no obvious correlation among the three quantities measured at similar locations within the tissues. The distribution of ATP corresponded well with the histological structure of larger spheroids; values were low in the necrotic center and high in the viable rim of these cell aggregates.

Cell and Environment Interactions in Tumor Microregions: The Multicell Spheroid Model

Abnormal vascularization of malignant tumors is associated with the development of microregions of heterogeneous cells and environments. Experimental models such as multicell spheroids and a variety of new techniques are being used to determine the characteristics of these microregions and to study the interactions of the cells and microenvironments. The special cellular microecology of tumors influences responsiveness to therapeutic agents and has implications for future directions in cancer research.

Multicellular spheroids. A review on cellular aggregates in cancer research.

Cellular aggregates have been used in developmental biology and in experimental cancer research for several decades. Spherical aggregates of malignant cells, i.e. multicellular tumor spheroids, may serve as in vitro models of tumor microregions and of an early, avascular stage of tumor growth. The similarities between the original tumor and the respective spheroids include volume growth kinetics, cellular heterogeneity, e.g. the induction of proliferation gradients and quiescence, as well as differentiation characteristics, such as the development of specific histological structures or the expression of antigens. Research using cell aggregates has been focussed on mechanisms involved in the control of proliferation, invasion and metastasis. Immunological studies with spheroids have resulted in the characterization of defense cells which are responsible for specific host-versus-tumor reactions. The vast majority of investigations on spheroids concerns the simulation of therapy with regard to various treatment modalities, combination treatments and systematic analyses of using various endpoints in predictive assays. Only a few pathophysiological studies on the interrelationship among tumor-specific micromilieu, cellular metabolism, proliferative status, and cellular viability have been undertaken with the spheroid model up to now. Since these studies are indicative of a large influence of the cellular microenvironment on basic biological properties of cancer cells, investigations of these epigenetic mechanisms should be intensified in future research on cell aggregates. Similarly, the molecular basis of the biological peculiarities found in malignant cells grown as three-dimensional aggregates has to be investigated more intensively.