Overview of Methods for Overcoming Hindrance to Drug Delivery to Tumors, with Special Attention to Tumor Interstitial Fluid.

Every drug used to treat cancer (chemotherapeutics, immunological, monoclonal antibodies, nanoparticles, radionuclides) must reach the targeted cells through the tumor environment at adequate concentrations, in order to exert their cell-killing effects. For any of these agents to reach the goal cells, they must overcome a number of impediments created by the tumor microenvironment (TME), beginning with tumor interstitial fluid pressure (TIFP), and a multifactorial increase in composition of the extracellular matrix (ECM). A primary modifier of TME is hypoxia, which increases the production of growth factors, such as vascular endothelial growth factor and platelet-derived growth factor. These growth factors released by both tumor cells and bone marrow recruited myeloid cells form abnormal vasculature characterized by vessels that are tortuous and more permeable. Increased leakiness combined with increased inflammatory byproducts accumulates fluid within the tumor mass (tumor interstitial fluid), ultimately creating an increased pressure (TIFP). Fibroblasts are also up-regulated by the TME, and deposit fibers that further augment the density of the ECM, thus, further worsening the TIFP. Increased TIFP with the ECM are the major obstacles to adequate drug delivery. By decreasing TIFP and ECM density, we can expect an associated rise in drug concentration within the tumor itself. In this overview, we will describe all the methods (drugs, nutraceuticals, and physical methods of treatment) able to lower TIFP and to modify ECM used for increasing drug concentration within the tumor tissue.

Tumor interstitial fluid: proteomic determination as a possible source of biomarkers.

Tumor interstitial fluid (TIF) is formed largely by an imbalance between the forces that govern the filtration of liquid between the luminal and abluminal parts of tumor neo-vessels. TIF is a dynamic solution that varies according to tumor type, and is generally rich in proteins, lipids, and various enzyme-derived substances. These enzyme-derived substances can have important roles as both regulatory and inflammatory factors. Furthermore, the oncotic pressure caused by the presence of these proteins and peptides in TIF leads to a proinflammatory condition in which macrophages produce cytokines such as Interleukins 1 and 6. With the recent advent of proteomics, TIF has been studied extensively and can be used as a source of potential biomarkers for cancer, including breast, ovarian, and head and neck cancer. In the present review, we discuss the process of TIF formation, its composition, the effects of its accumulation, the methods of sampling, and the proteomic analyses performed on it, which make TIF a valuable tool in monitoring several cancer types.

Tumor regression with a combination of drugs interfering with the tumor metabolism: efficacy of hydroxycitrate, lipoic acid and capsaicin.

Cellular metabolic alterations are now well described as implicated in cancer and some strategies are currently developed to target these different pathways. In previous papers, we demonstrated that a combination of molecules (namely alpha-lipoic acid and hydroxycitrate, i.e. Metabloc™) targeting the cancer metabolism markedly decreased tumor cell growth in mice. In this work, we demonstrate that the addition of capsaicin further delays tumor growth in mice in a dose dependant manner. This is true for the three animal model tested: lung (LLC) cancer, bladder cancer (MBT-2) and melanoma B16F10. There was no apparent side effect of this ternary combination. The addition of a fourth drug (octreotide) is even more effective resulting in tumor regression in mice bearing LLC cancer. These four compounds are all known to target the cellular metabolism not its DNA. The efficacy, the apparent lack of toxicity, the long clinical track records of these medications in human medicine, all points toward the need for a clinical trial. The dramatic efficacy of treatment suggests that cancer may simply be a disease of dysregulated cellular metabolism.

Update on the challenges and recent advances in cancer immunotherapy.

This overview provides an analysis of some of the immunotherapies currently in use and under investigation, with a special focus on the tumor microenvironment, which we believe is a major factor responsible for the general failure of immunotherapy to date. It is our expectation that combining immunotherapy with methods of altering the tumor microenvironment and targeting regulatory T cells and myeloid cells will yield favorable results.

Tumor interstitial fluid as modulator of cancer inflammation, thrombosis, immunity and angiogenesis.

Tumor interstitial fluid (TIF) is a watery phase that accumulates inside the tumor interstitium. Its genesis and fate depend on various factors, namely tumor type, metabolic state of the tumor, expression of vascular endothelial growth factor, and absence of lymphatic system. For almost 30 years TIF remained a neglected entity until it was demonstrated that TIF, and in particular its high pressure, constitutes an important obstacle to drug delivery and immunotherapy. The present review not only summarizes the abundant literature on the processes of TIF genesis and on its effects on therapy but it also presents data that, in our opinion, point towards what is perhaps the real physiological purpose of TIF: a primitive means of providing nourishment, oxygen, cytokines and matrikines to tumor cells that furthermore promotes the invasion of the normal surrounding tissue and passive metastatization through lymphatics. It is also an inducer of inflammation through increased osmolarity due to albumin loss. Recently, a role for TIF as a possible source of biomarkers has also been suggested.

Adding a combination of hydroxycitrate and lipoic acid (METABLOC™) to chemotherapy improves effectiveness against tumor development: experimental results and case report.

Altered metabolism of cancer first highlighted by Otto Warburg has a long history. Although ignored for a considerable amount of time, it is now receiving substantial attention. We recently published results obtained with a combination of two drugs, lipoic acid and hydroxycitrate, targeting metabolic enzymes particularly affected in cancer: ATP citrate lyase and pyruvate dehydrogenase kinase. This treatment was as efficient as chemotherapy in the three mouse cancer models that were tested. In this work, we asked if our drug combination could be used in conjunction with standard cytotoxic chemotherapy, in particular cisplatin, to improve basic protocol efficacy. A combination of lipoic acid and hydroxycitrate was administered to mice implanted with syngeneic cancer cells, LL/2 lung carcinoma and MBT-2 bladder carcinoma, concommitantly with classical chemotherapy (cisplatin or methotrexate). We demonstrate that the triple combination lipoic acid + hydroxycitrate + cisplatin or methotrexate is more efficient than cisplatin or methotrexate used individually or the combination of lipoic acid and hydroxycitrate administered alone. Of particular note are the results obtained in the treatment of an 80 year-old female who presented with ductal adenocarcinoma of the pancreas accompanied by liver metastases. A treatment course using gemcitabine plus α-lipoic acid and hydroxycitrate gave highly promising results. The in vivo data, coupled with the case study results, suggest a possible advantage in using a treatment targeted at cancer metabolism in association with classical chemotherapy.

Screening of well-established drugs targeting cancer metabolism: reproducibility of the efficacy of a highly effective drug combination in mice.

Alterations in metabolic pathways are known to characterize cancer. In order to suppress cancer growth, however, multiple proteins involved in these pathways have to be targeted simultaneously. We have developed a screening method to assess the best drug combination for cancer treatment based on targeting several factors implicated in tumor specific metabolism. Following a review of the literature, we identified those enzymes known to be deregulated in cancer and established a list of sixty-two drugs targeting them. These molecules are used routinely in clinical settings for diseases other than cancer. We screened a first library in vitro against four cell lines and then evaluated the most promising binary combinations in vivo against three murine syngeneic cancer models, (LL/2, Lewis lung carcinoma; B16-F10, melanoma; and MBT-2, bladder cancer). The optimum result was obtained using a combination of α-lipoic acid and hydroxycitrate (METABLOC(TM)). In this study, a third agent was added by in vivo evaluation of a large number of combinations. The addition of octreotide strongly reduced tumor development (T/C% value of 30.2 to 34.5%; P < 0.001) in the same models and prolonged animal survival (P < 0.001) as compared to cisplatin. These results were confirmed in a different laboratory setting using a human xenograft model (NCI-H69, small cell lung cancer). None of these three molecules are known to target DNA. The effectiveness of this combination in several animal models, as well as the low toxicity of these inexpensive drugs, emphasizes the necessity of rapidly setting up a clinical trial.

Cytotoxic Activity of Peripheral Blood Mononuclear Leukocytes, Activated by Interleukin-2/ß-Cyclodextrin Nanocomposition against Androgen Receptor-Negative Prostate Cancers.

Nanocomposition comprised of interleukin-2 in suboptimal noneffective concentration and ß-cyclodextrin was studied in vitro. This preparation as well as interleukin-2 in optimal concentration was shown to increase natural killer activity to K-562 cells and cytotoxicity of activated peripheral blood mononuclear cells (PBMCs) against PC-3 and DU 145 cells. At the same time ß-cyclodextrin or interleukin-2 in equimolar concentrations did not influence the spontaneous killer activity of PBMC. This combination of cyclodextrin + interleukin-2 led to the decrease of interleukin-2 effective concentration by an order. This phenomenon could be explained by cyclodextrins ability to promote the formation of nanoparticles with drugs, which results in enhancing their water solubility and bioavailability. Besides, interleukine-2/ß-cyclodextrin nanocomposition as opposed to interleukin-2 alone led to increasing the number of not only lymphocytes, but also macrophages contained in activated PBMC population. Application of low concentration of interleukin-2 allowing for good clinical efficiency may significantly mitigate the side effects of the drug and enable to develop adoption of immunotherapy for patients with androgen-resistant prostate cancer.

Is it necessary to deplete the lymphokine activated killers' populations of CD4+CD25+ lymphocytes? Regulatory Foxp3-positive T cells within lymphokine activated killers.

LAKs are used in cancer therapy. A common argument against LAK therapy is the probability of the activation of Tregs by IL-2 alongside with NK cells. In order to evaluate negative impact of Tregs, contents of Foxp3(+)CD4(+)CD25(+) Tregs and their influence on LAKs' cytotoxic activity in the samples of healthy volunteers' PBMCs cultured with or without IL-2 were determined. A marked increase in the proportion of the CD4(+)CD25(+) T cells in the most of the PBMCs cultured with IL-2 was evident. Surprisingly, the contents of Foxp3(+)CD4(+)CD25(+) Tregs showed variable response to IL-2 in blood samples of different people. Their proportions increased, remained on the same level or even decreased compared to PBMCs cultured without exogenous IL-2. Different methods of Treg isolation were compared regarding the purities of the resulting T-cell populations and their enrichment in the Foxp3-expressing Tregs, as well as their viability. Neither of the kits applied provided any enrichment in Foxp3-level in the purified cells compared to the CD4(+)CD25(+) T cells gated on applying flow-cytometric cell analysis in the non-separated LAKs. Moreover, the addition of the purified supposedly Tregs to LAKs never inhibited their cytotoxic reactions, even then they were added in great non-physiological excess. In some of the blood samples, there have been detected another non-identified type of the Foxp3-expressing cells, which lacked CD25, as well as CD4. They may be a new type of regulatory cells, which has not been described before.

Review. Current role and future perspectives of hyperthermia for prostate cancer treatment.

Prostate cancer is the second most common cause of death in men. Between 10%-60% of patients experience biochemical recurrence associated with treatment failure. No consensus on optimal therapy for advanced prostate cancer exists and even though hormonal therapy and radiotherapy are standards, different groups have included hyperthermia associated with radiotherapy to increase its efficacy. We have tried to analyze these studies to evaluate if this association can ameliorate the therapy in the advanced disease state. From the analysis of these studies, it appears that no amelioration can be obtained adding hyperthermia and the principal cause of this failure appears to be due to the incorrect timing of hyperthermia application and probably the abrogation of androgen receptor expression in androgen-dependent cells.

Hyperthermia and immunity. A brief overview.

After many years, hyperthermia (HT) is experiencing a new resurgence as seen by the positive results of many randomized trials all over the world. Tumour immunity similarly is suggested as the fourth modality of therapy for metastatic tumours from renal carcinoma and melanoma. An overwhelming amount of data from animal models and human patients indicate that whole body and locoregional hyperthermia exerts many biological and therapeutic effects on immune competent cells and cytokines. Among these effects, hyperthermia has recently been demonstrated to enhance the antigen presentation and consequently the activity of dendritic cells. This improvement is obtained through several mechanisms: a) increased lymphocyte recruitment and trafficking into the tumour area; b) increased immunogenicity of heat treated tumour cells; and c) increased production of the heat-shock proteins and costimulatory molecules. The effects and mechanisms of HT on immunity, lymphocyte recruitment and dendritic cell stimulation by heat shock proteins are reviewed here. Moreover the use of HT as an innate immunity booster in association with biological response modifiers is suggested.

Stem cell recruitment and liver de-differentiation in MMTV-neu (ErbB-2) transgenic mice.

The liver of tumor-bearing hosts manifests fetal phenotypes. We investigated the expression of differentiation markers on the liver in MMTV-neu (ErbB-2) transgenic mice, in the period from incipient neoangiogenesis to lung metastatization. We report AFP expression by hepatocytes in all lobular zones, CD34 cell arrest and subsequent hemopoiesis in periportal and mid-zone areas, oval-like cells (CD34+, CK19+, AFP+) and ductular reaction in portal tracts, portal CK19+ and GGT+ hepatoblast-like cells, and midzonal large dysplastic hepatocytes. We hypothesize that CD34 cells are recruited by the tumor from the marrow for angiogenic purposes and that their differentiation in the liver is influenced by altered liver microenvironment(s). AFP may act as a growth factor and biological response modifier for these cells and for the tumor. Dysplasia might be enhanced by metabolic stress. We conclude that the liver differentiation potential is lobular-zone-dependent and that the risk for eventually developing a pre-malignant lesion is not negligible.

Tumor microenvironment and hemorheological abnormalities.

This article reviews the various mechanisms in a malignant tumor that lead to hypoxia, production of tumor interstitial fluid, and rheological changes in its microenvironment. In addition, the associated procoagulant effects are described. The latter phenomenon is the result of complex and dynamic interplay among tumor cells, endothelial cells, circulating blood cells, and angiogenic, clotting, and hemorheological factors. The implications of these abnormalities on therapeutic approach are discussed.