TUMOR MICROENVIRONMENT AND DRUG RESISTANCE
The tumor vasculature is a critical regulator of the delivery of nutrients to tumor cells and of the removal of products of metabolism and consequently of tumor growth. Although tumor cells release factors that stimulate angiogenesis, tumor vasculature often lacks an organized structure. As cancer cells proliferate, blood vessels may become separated by longer distances than in normal tissues, and raised interstitial fluid pressure (IFP) and a dense extracellular matrix may compress blood vessels and contribute to irregular blood flow.1,2 Consequently, there is a limited distribution of nutrients, including oxygen to cells located distally from functional blood vessels. There may also be a buildup of cellular breakdown products in such regions including lactic and carbonic acids, leading to an acidic microenvironment in tumors (Fig. 1). The abnormal vascular structure and function of solid tumors also limit the delivery of anticancer drugs.2–4
For a systemically administered anticancer drug to kill a high proportion of cancer cells in a solid tumor, it must cross blood vessel walls and penetrate into tumor tissue. However, the distribution of many drugs within tumors is heterogeneous, so that only a proportion of the target tumor cells are exposed to a potentially lethal concentration of the cytotoxic agent: regions distal to blood vessels receive lower amounts of drug than do those cells located proximal to tumor blood vessels. Within nutrient-deprived tumor regions, the rate of tumor cell proliferation is relatively low,5,6 and slowly proliferating cells are resistant to most currently used anticancer drugs, including many targeted agents. Both of these effects contribute to the relative resistance to anticancer drugs of cells located distal to functional blood vessels.
The convoluted vasculature within tumors results in regions where the oxygen concentration falls to zero.7 The distance from functional blood vessels to hypoxic regions will depend on the rate of oxygen consumption by the tumor cells but is typically at a distance greater than 70 μm.8 Cells in chronically hypoxic regions may be viable, but they are often adjacent to region of necrosis and are probably destined to die in the absence of treatment. Acute hypoxia is also common in tumors and results from intermittent blood flow through tumor vessels.9,10
Tumor hypoxia is an important mediator of resistance to cancer treatment. Cellular sensitivity to radiation depends on the generation of free radicals in the presence of oxygen, so that hypoxic cells are relatively radioresistant. Hypoxic cells may also be resistant to many anticancer drugs because (i) drugs are unlikely to be delivered in toxic concentrations to hypoxic regions; (ii) low concentrations of nutrient metabolites in hypoxic regions inhibit cancer cell proliferation, and slowly proliferating cells are resistant to cycle-active drugs2; (iii) tumor hypoxia is linked to loss of the p53 tumor suppressor protein that may result in loss of apoptotic ability and to increased angiogenesis and invasiveness11,12; (iv) hypoxic cells can down-regulate expression of DNA topoisomerase II, so that drugs such as doxorubicin and etoposide that target this protein will be inefficient13; (v) hypoxia up-regulates genes involved in drug resistance, including MDR genes encoding the drug export pump P-glycoprotein14; and (vi) hypoxia can induce autophagy, which is a survival mechanism of stressed cells.15,16
The extracellular pH in solid tumors is often lower than in normal tissues, because of a high rate of glycolysis with production of lactic and carbonic acids and poor clearance of these acids.17,18 Hypoxia also up-regulates the expression and activity of carbonic anhydrase, which leads to enhanced extracellular acidification.19 The acidic extracellular environment causes basic anticancer drugs (such as doxorubicin) to be charged, resulting in poorer uptake into cells because it is the uncharged form that diffuses across the cell membrane.
The strong pH gradient between the cytoplasm of tumor cells and acidic intracellular organelles such as endosomes and lysosomes20,21 may also be involved in resistance to anticancer drugs. Many anticancer agents are weak lipophilic bases (e.g., doxorubicin, mitoxantrone), and they will accumulate in acidic organelles. When drugs are sequestered in the organelles of tumor cells, they become less effective because less drug is available to enter the nucleus to exert cytotoxic effects18; sequestration of drugs in these compartments will also lead to less drug being available to diffuse to cells more distant from functional blood vessels (Fig. 2). As well as being sites of sequestration of basic drugs, acidic endosomes are central to the process of autophagy, discussed below as a cause of drug resistance.
We have shown that agents that raise endosomal pH might decrease endosomal sequestration of basic anticancer drugs and thereby modify their toxicity and tissue penetration.22 Increasing endosomal pH might be achieved by concomitant use of other basic compounds (such as chloroquine) or by using inhibitors of the proton pump that maintain endosomal pH at a low value (Fig. 2). This pump is related to that which maintains acidity in the stomach and may be inhibited by proton pump inhibitors (PPIs), which are used clinically to reduce gastric acidity. We and others have obtained evidence that PPIs may enhance the effects of chemotherapy in animal models.23–27
Autophagy is a cellular mechanism used to digest old or damaged cellular components into component residues, which may be recycled to generate essential macromolecules. All cells undergo autophagy, but it is up-regulated in stressed cells such as those with nutrient or growth factor depletion and hypoxia; such stress is common in nutrient-deprived regions of solid tumors, and autophagy colocalizes with hypoxia in tumors.16,28 Autophagy involves the formation of autophagosomes, which have a double membrane enclosing cytoplasmic cellular components; these then fuse with lysosomes to produce mature autolysosomes in which cellular proteins are degraded by cathepsins29–31 (Fig. 3).
A series of autophagy-related proteins (known as ATGs) are responsible for the induction and regulation of autophagy,32 and some of them are used as markers of autophagy that can be quantified in Western blots or by immunohistochemistry (IHC) applied to tumor sections. The human form of ATG8 is microtubule-associated protein light-chain 3B (LC3B), which exists in a cytosolic form as LC3B-I. Upon activation of autophagy, LC3B-I is cleaved and modified to LC3B-II, which then binds to the membrane of the autophagosome. Because of its direct association with the autophagosome, LC3B-II has been used widely as a marker of autophagy in mammalian models.33,34 However, LC3B-II can undergo turnover after fusion of the lysosome,33,34 so that increase in membrane-bound LC3B-II can be due to either increased LC3-I processing from activation of autophagy or a buildup of membrane-bound LC3B-II following inhibition of lysosomal fusion. Use of antibodies to determine activation or inhibition of autophagy using LC3B isoforms is difficult if only LC3B-II is used. An additional autophagy marker, p62/SQSTM1 (p62), is recruited with LC3B-II to autophagosomes but, unlike LC3B-II, is degraded within the mature autolysosome.34,35 Thus, observation of increased p62 is indicative of a buildup of the protein because of inhibition of lysosomal fusion to the autophagosome, that is, to inhibition of autophagy.
Autophagy is prognostic of poor outcome in multiple tumor types, including cancers of the breast, lung, colon, and melanoma.36–39 High levels of autophagy have been associated with resistance to systemic therapy in several preclinical and clinical models, presumably because autophagy facilitates survival of stressed or damaged cells through recycling of cellular breakdown products.40 We have shown that treatment of cancer cells in vitro and of solid tumors in mice with a wide variety of anticancer agents induces autophagy, suggesting that it is a common survival mechanism for drug-damaged cells, especially those where autophagy is already up-regulated because of nutrient deprivation and therefore an important cause of drug resistance.
Drug Distribution in Solid Tumors
Drugs exit blood vessels and penetrate into tissues by convection and/or diffusion. Convection depends on pressure gradients between the vascular space and the interstitial space, vessel permeability and the surface area for exchange, and the volume and structure of the extracellular matrix; it is most important for transport of large molecules.41 Drug diffusion is determined by concentration gradients and by the size and charge of the molecules and is the dominant mechanism for drugs of smaller molecular weight.42 Diffusion of drugs is also limited by binding in tissue or by their rapid metabolism once they have extravasated. An important determinant of drug distribution within tissues is the half-life of the drug in the circulation; drugs that have a long half-life have a better opportunity to achieve equilibrium within the tumor microenvironment.43
Drug distribution can be evaluated in cell culture systems and in experimental solid tumors. In vitro multicellular models include tumor spheroids and multilayered cell cultures (MCCs).44–47 Spheroids develop hypoxic areas as well as central areas of necrosis once they grow to ∼500 μm in diameter.48 Drug distribution in spheroids can be studied for fluorescent drugs or by using autoradiography to determine the distribution of labeled drugs.49,50 Experiments using spheroids have shown steep gradients of drug concentration from the surface for doxorubicin and methotrexate.45,48 Multilayered cell cultures are grown on collagen-coated microporous membranes: the rate of drug penetration can be evaluated by adding a drug on one side of the MCC and measuring its concentration on the other as a function of time.51 This method has been used to demonstrate slow tissue penetration of a range of clinically used chemotherapeutic agents, including etoposide, gemcitabine, paclitaxel, and vinblastine46,52 (Fig. 4).
Drug distribution can also be studied in animal models. Growth of tumors in window chambers allows for direct observation of tumor microcirculation.53,54 Tumors can be excised at different times after drug treatment of animals bearing transplanted tumors, including human tumor xenografts, and tissue sections cut and processed for IHC. This analysis allows quantification of fluorescent drugs in relation to blood vessels or regions of hypoxia (defined by a marker which identifies hypoxic cells, such as EF5 or pimonidazole), and the technique can be applied to biopsies of human tumors.3,55 We have used this method to quantify the distribution of the fluorescent drugs doxorubicin, mitoxantrone, and topotecan in relation to blood vessels and have shown steep gradients of decreasing drug concentration with increasing distance from tumor blood vessels and low penetration of drugs into hypoxic areas.56,57 For example, doxorubicin intensity decreases to half at approximately 40 to 50 μm from the nearest blood vessels such that many viable cells are not exposed to detectable concentrations of drug after a single injection (Fig. 5A).
Most anticancer drugs are nonfluorescent, so their distribution within tumor tissues is difficult to assess. Fluorescently tagged antibodies can recognize some drugs, such as the monoclonal antibodies cetuximab and trastuzumab; these agents have an initial poor distribution, but equilibrium is then reached throughout the tumor, most likely because of their long half-lives in the circulation.58 The distribution of activity of other drugs can be evaluated by molecular markers of drug effect, using fluorescence-tagged antibodies that recognize cell proliferation (Ki67), antibodies that mark cell death or apoptosis (e.g., activated caspase 3 or 6), and markers of DNA damage such as γH2AX.59 We have validated the use of these biomarkers to study the distribution of activity of anticancer agents in solid tumors and have used quantitative IHC to demonstrate decreasing levels of activity with increasing distance from tumor blood vessels for doxorubicin, docetaxel,27,59 and paclitaxel (Figs. 5B, D).
Repopulation of Tumors Between Treatments
Repopulation of surviving cancer cells between daily fractions during a course of radiotherapy has long been recognized as a cause of resistance. Similar repopulation occurs in the longer intervals between courses of chemotherapy; in normal tissues, this allows recovery (e.g., of bone marrow), but in tumors, it may be a cause of treatment failure.60,61 Our recent work has used dual labeling of hypoxic cells with EF5 and pimonidazole, with a variable interval between applications of these markers, to determine the fate of tumor cells that were hypoxic at the time of treatment. For reasons discussed previously, hypoxic tumor cells are resistant to chemotherapy. We demonstrated that hypoxic cells that were destined to die in the absence of treatment can survive and repopulate the tumor after chemotherapy62
STRATEGIES TO IMPROVE THERAPY
Most studies of drug resistance have concentrated on the multiple molecular causes of resistance in individual cancer cells. Although these are important, even drug-sensitive cells can respond to treatment only if they are exposed to an adequate drug concentration. It is evident from the above that limited distribution within solid tumors limits exposure of tumor cells to many drugs, and limited drug distribution is an important and neglected cause of drug resistance. A variety of strategies are being researched to improve or complement the limited distribution of commonly used anticancer drugs, and these are described in the following sections.
Improving Drug Distribution
Several strategies might lead to improved drug distribution within solid tumors. Giving drugs by continuous infusion to maintain levels in blood vessels can achieve this but will also increase delivery to normal tissues and will not necessarily improve the therapeutic index.63 Many investigators have delivered drugs in liposomes or other nanoparticles, with the expectation that they may selectively penetrate blood vessels in tumors and then release drug over a prolonged period to increase tumor distribution,64 but evidence for improved therapeutic effects is minimal. Any strategy that leads to decreased drug uptake in cells close to blood vessels will increase penetration to more distal cells; for example, we have shown improved distribution of doxorubicin by inhibiting its sequestration in acidic endosomes using PPIs,26 although the effect is modest and probably not the major mechanism by which antitumor activity is increased by these agents.
High IFP may have an adverse effect on treatment because it may cause vascular compression and inadequate drug delivery through reduced convection. Human pancreatic tumors are extremely resistant to systemic cancer therapy, and there is evidence that this is due to high IFP or to solid tissue stress due to elements of the extracellular matrix preventing the delivery of drugs to constituent cells. Reduction of IFP using hyaluronidase or of tumor-associated stroma tissue by other agents has been reported to improve delivery of chemotherapy in experimental models.65,66 A recent study showed that imatinib, a molecular targeting drug, was able to reduce tumor IFP in melanoma and allowed delivery of more doxorubicin into tumor tissue. Combined treatment inhibited tumor growth and induced apoptosis of tumor cells.67
Agents that inhibit endosomal acidification, including (hydroxy)chloroquine and PPIs, can suppress autophagy, which is a potential survival mechanism following chemotherapy, especially for nutrient-deprived cells. An Italian group reported the use of PPIs to sensitize cancer cells to various chemotherapeutic agents. Multiple mechanisms are likely involved but appear to relate to changes in acidity in both intracellular and extracellular compartments of tumor cells. Proton pump inhibitors can inhibit autophagy probably because fusion of autophagosomes with acidic endosomes is central to the process. We have confirmed that the PPI pantoprazole inhibits autophagy in vitro and in vivo and that it enhances activity of docetaxel against human tumor xenografts, with improved distribution of activity, as determined by the biomarkers γH2AX and cleaved caspases throughout the tumors.27 We showed marked effects of docetaxel and some other anticancer drugs to up-regulate autophagy as indicated by increased levels of LC3B and reduced levels of p62 in all tumor regions (Fig. 6), with opposite effects indicating inhibition of autophagy, when chemotherapy is combined with pantoprazole. We obtained further evidence for this being the main mechanism of action by using autophagy-deficient cells generated by shRNA knockdown of the autophagy proteins ATG7 and BECLIN1 or both.27 Several other studies have shown that PPIs such as omeprazole, esomeprazole, and pantoprazole have activity against human hematopoietic and solid tumors; they may revert chemoresistance in drug-resistant tumors and directly induce killing of tumor cells.24,68,69 Our unpublished data show that a wide spectrum of anticancer drugs up-regulate autophagy in cultured tumor cells and that this can be inhibited by PPIs. Growing evidence suggests that the major mechanism by which PPIs enhance the effects of chemotherapy is by inhibition of autophagy27,70
Hypoxia-activated prodrugs (HAPs) are administered in an inactive form and are activated in the absence of oxygen. Hypoxic regions within solid tumors can be identified by IHC, using markers of hypoxia such as pimonidazole and EF5. We have shown that the HAP, TH-302, was able to increase expression of γH2AX and cleaved caspases in hypoxic areas of human tumor xenografts.71 The anticancer drugs docetaxel and doxorubicin had minimal activity in hypoxic regions, but the combination of TH-302 and chemotherapy resulted in increased expression of γH2AX and cleaved caspases, compared with use of either drug alone, in regions both proximal and distal to blood vessels. Thus, the distributions of activity of the chemotherapy drugs and of the HAP TH-302 complement each other, although there appear to be additional effects to enhance overall activity.62 The combination of gemcitabine and TH-302 has given encouraging results in a phase II trial for human pancreatic cancer,72 and a phase III trial is in progress.
Inhibition of Tumor Cell Repopulation
Therapeutic index might be improved by inhibiting selectively the repopulation of tumor cells that have the ability to regenerate the tumor between courses of chemotherapy, without inhibiting repopulation of critical normal tissues. This has been attempted in model systems using cytostatic inhibitors such as hormonal agents in breast cancer and inhibitors of growth factors,73,74 but effects on outcome have been modest. As indicated above, the poor distribution of drugs in solid tumors leads to survival of hypoxic and other poorly nourished cells, which might then repopulate the tumor. By administering pimonidazole and EF5 sequentially and labeling with the proliferation marker Ki67, we have been able to follow reoxygenation and proliferation of tumor cells that were hypoxic at the time of administration of chemotherapy.62 We demonstrated that chemotherapy (with doxorubicin or docetaxel) induced repopulation and reoxygenation of tumor cells that were previously hypoxic. Moreover, these processes were inhibited by the HAP TH-302, indicating another potential mechanism whereby combined targeting of well-nourished cells with chemotherapy, and hypoxic cells with HAPs, might improve therapeutic effects62 (Fig. 7).
The effectiveness of anticancer drugs is impaired by their limited distribution within tumors. Agents that modify or complement the activity of anticancer drugs have the ability to improve treatment outcomes. Modification of autophagy to increase the toxic activity of drugs within tumors and use of hypoxia-activated prodrugs hold particular promise to improve the effectiveness of conventional chemotherapy.
The authors thank all members of the Pathology Research Program (PRP) and the Advanced Optical Microscopy Facility.
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