Dietmar W. Siemann - US grants

Our hypothesis is that a tumor can be considered as an entity composed of a variety of cell subpopulations. Each subpopuIation may have its own inherent properties and intrinsic sensitivities to therapies. These different sensitivities can arise from environmental or genetic factors or both. The overall tumor response to anticancer therapies then is a reflection of the responses of the individual cell subpopulations. The central goal of this research proposal continues to be the investigation of the various critical cell subpopulations comprising solid tumors. We will use cell separation techniques including centrifugal elutriation and fluorescence activated cell sorting to isolate and characterize cell subpopulations dispersed from rodent tumors, human tumor xenografts or clinical samples. Emphasis will be placed on determining the biological characteristics of hypoxic cells. There is evidence to suggest that tumors may contain cells which may be chronically or transiently hypoxic. Such cells would result from different mechanisms, have different characteristics, and have different implication for therapies. We plan to probe various rodent and human tumor models for the presence of such cells using tracer dyes combined with histological and flow cytometric techniques as well as radiolabeled sensitizers. Quiescent (Q) cells may represent another cell subpopulation of considerable relevance in clinical anticancer therapy. Experiments are proposed to study the response of such cell to different chemotherapeutic agents. Q cells in rodent tumor models will be characterized by acridin orange (AO) staining and flow cytometry analysis and isolated by centrifugal elutriation. In human tumor cells, FlTC conjugated anti-Ki-67 and anti-p105 antibodies and flow cytometry will be used to determine the percentage of proliferating cells. In parallel with the AO staining and Ki-67 or plO5 analyses, 3H-TdR labelling will be performed. Experiments extending Q cell determinations to human tumor biopsies also are proposed. Since changes in the quiescent cell population, if they occur during the course of therapy, could have a major impact on therapy, it is also our goal to attempt to establish techniques for measuring Q cell fractions in treated tumors. Finally, experiments are designed to gain a better understanding of the biological basis and underlying mechanisms for tumor response to altered fractionation schemes. Ultimately we hope that these approaches can be reliably developed to an extent where the use of human tumor biopsy material may lead to radiotherapy treatments more tailored to the individual patient.

Interactions of various nitroheterocyclic radiation sensitizers with cancer chemotherapeutic agents and radiation used in combination will be studied in an in vivo lung tumor model. Experiments are proposed to determine the effect on tumor cells growing in the lung of adding a sensitizer to a protocol combining the nitrosourea CCNU with localized radiation or the alkylating chemotherapeutic agent cyclophosphamide. Treatment efficacy will be assessed using endpoints of clonogenic cell survival, tumor regrowth delay and tumor-free animal survival. The influence that the inclusion of a sensitizer in such protocols has on secondary ovarian and renal metastases arising from lung tumors also will be established. Both the incidence of metastases to the ovaries and kidneys and their response to the treatment regimen will be measured. The nature of the interaction between such combined modality therapies will be determined through isoeffect plot (isobologram) analysis. It also is our objective to elucidate the role of pharmacokinetic changes (using HPLC analysis) in the combined modality protocols. The potential therapeutic benefit which may be achieved through the addition of a sensitizer to the different treatment regimen will be determined by measuring tumor responses as well as the early and late effects on critical normal tissues. Both tumor and normal tissue responses also will be studied under conditions where sensitizers are administered such that sensitizer pharmacokinetics achievable in human plasma are mimicked. Those treatment protocols giving rise to the largest therapeutic gains will be evaluated in xenografts of human lung cancer. These investigations should yield information which will improve our understanding of combined modality therapies and may guide the efforts for the most effective use of combinations of sensitizers, chemotherapeutic agents and radiation.

The overall objective of this research program is to determine the importance of various tumor cell subpopulations in predicting a tumor's response to single and combined modality therapies. Cell subpopulations will be derived from solid KHT, RIF-1 or EMT6/Ro tumors using centrifugal elutriation. Experiments are proposed to determine the proportion and cell cycle location of quiescent (Q) cells and to measure growth imbalance in these solid tumor models using acridine orange (AO) staining and flow cytometric (FCM) analysis. The location, with respect to cell cycle phase and proliferation state, of oxygen-deficient (hypoxic) tumor cells will be characterized using radiation cell survival curve analysis, combinations of centrifugal elutriation plus viable cell sorting (Hoechst 33342) and the identification of hypoxic cells through the use of labelled hypoxic cell sensitizers. In situ sensitivity differences of various tumor cell subpopulations (including hypoxic and quiescent cells) will also be determined. It is further an objective to assess which subpopulations are most affected by radiation, sensitizers and selected chemotherapeutic agents. Possible interactions of sensitizers, drugs and radiation in terms of their effects on the total tumor as well as specific subpopulations will be evaluated. These studies should allow the development of mechanisms of interactions for the different combined modality treatments. Experiments aimed at determining the response of tumors and their subpopulations to different fractionated dose regimen also will be performed. In particular the effects of fractionated therapies on cell cycle redistribution and quiescent cell recruitment will be measured. These investigations should produce information which will improve the understanding of the role played by various cell subpopulations in determining the overall response of tumors to therapy and which may guide the efforts for the most effective use of combinations of radiation, chemotherapeutic agents and sensitizers.

The central focus of this grant application is to utilize pathophysiological manipulations to attempt to overcome the radiation resistance associated with transient (acute) and diffusion-limited (chronic) tumor hypoxia. Investigations will evaluate the effects of changes in hemoglobin (Hb) affinity for oxygen (O2) and/or tumor blood flow on tumor response to radiation. Hb affinity will be altered by the direct administration of agents which shift the Hb-02 dissociation curve or the transfusion of biochemically modified erythrocytes into tumor-bearing recipients. Tumor blood flow will be manipulated through the administration of the agents flunarizine and nicotinamide. Changes in tumor blood flow will be monitored using a variety of techniques including laser Doppler flowmetry, and the relationship between blood flow changes and radiation sensitivity will be established. To study whether specific manipulations of tumor oxygenation are producing the desired effects at the microregional level, intravascular HbO2 saturations distributions will be determined cryospectrophotometrically. Additionally, dual-staining procedures with Hoechst 33342 and DiOC 7(3) will be used to determine the relative fraction of vessels involved in transient perfusion. These studies initially will utilize the rodent KHT and SCCVII tumor models since chronically hypoxic cells dominate the former while acutely hypoxic cells can be demonstrated in the latter. For comparison, more limited experiments in human ovarian tumor xenografts also are proposed. Xenograft studies are of particular interest given the recent. controversy concerning the activity of vasoactive -agents in human versus rodent tumor models. It also is our objective to evaluate in these tumor models the therapeutic potential of combining therapies directed at the different types of hypoxia. Those conditions leading to the greatest antitumor activity will be evaluated using fractionated dose radiotherapy protocols. These investigations should improve our understanding of tumor hypoxia and may have implications for strategies aimed at overcoming hypoxia-mediated treatment resistance by specifically targeting acute and chronically hypoxic cells.

DESCRIPTION (Applicant's Description) The aim of this grant application is to request partial travel support to allow domestic and foreign Junior Investigators to attend and participate in the Tenth International Conference on Chemical Modifiers of Cancer Treatment to be held in Clearwater, Florida, January 28-February 1, 1998. As in the previous nine conferences the central theme of this conference will be the use of chemical agents to modify the response of tumors and/or normal tissues to the cytoxic effects of radiotherapy of chemotherapy. The ultimate objective is to improve the therapeutic ratio. At the core of the Chemical Modifiers conferences is the complex tumor microenvironment which is likely to be both a major contributor to treatment failure and an opportunity for new therapeutic strategies. Topics addressed at the Tenth Chemical Modifiers Conference will include hypoxic cell sensitizers and cytotoxins, radiation protectors, angiogenesis and anti-angiogenic therapies, gene therapy approaches based on the tumor microenvironment, tumor physiology as a predictor for therapy outcome, techniques for assessing tumor hypoxia, in situ, manipulation of oxygen transport, etc. The conference will incorporate key note lectures, proffered abstracts coupled with two page handouts, and ample time for discussion. A publication of the proceedings of the conference will collate this information in a published reference. The Tenth International Conference on Chemical Modifiers of Cancer Treatment will bring together basic and clinical scientists from six continents. The conference is a true reflection of the concept of translational research as it will foster the two way exchange of information between the laboratory and the clinic and will facilitate progress toward gaining a better understanding of the underlying principles of cancer and developing improved cancer therapies.

tubulin; binding proteins; combination cancer therapy; neoplasm /cancer radiation therapy; neoplasm /cancer blood supply; nonhuman therapy evaluation; angiogenesis; colchicine; dosage; tissue /cell culture; immunocytochemistry; laboratory mouse; human tissue; histology;

DESCRIPTION (provided by applicant):Radiotherapy is the most important non-surgical treatment for cancer. Even so, large numbers of patients still fail at the local treatment site. New treatment strategies aimed at improving tumor response are therefore of high interest and the tumor vasculature, which is critical for the growth and survival of the neoplastic cell population, offers an attractive target. The goal of this grant proposal is to investigate approaches for optimally combining antiangiogenic strategies with localized radiation therapy in order to improve overall tumor response. Investigations are focused on interfering with two activators of angiogenesis (vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF)) and amplification of an endogenous suppressor (endostatin). The approach will be the delivery and transfer of cDNA coding for antisense VEGF/bFGF or endostatin using adeno-associated virus (AAV) or cationic liposomes. Experiments are designed to first characterize and optimize the proposed antiangiogenic approaches in tumor cells, fibroblasts and endothelial cells in vitro. These studies will include the examination of the effect of angiosuppression strategies on the paracrine communication between the various cell types and the determination of whether direct interactions occur between such strategies and radiation treatment. The in vivo efficacy of antiangiogenic treatments used in conjunction with radiation therapy will then be evaluated in xenograft models of AIDS associated Kaposi's Sarcoma and clear cell renal cell carcinoma. These models were chosen because we believe that these neoplasms' typical manifestation of extensive vascularization, coupled with their lack of satisfactory responses to traditional therapeutic interventions, make them excellent candidates for new therapeutic strategies such as antiangiogenic approaches. Both tumor response and critical normal tissue toxicities will be assessed to determine whether a therapeutic benefit can be achieved when angiosuppressive treatments and radiation are combined. We believe that these studies will provide essential insights into the therapeutic utility of employing antiangiogenic treatment strategies as adjuvants to radiotherapy.

DESCRIPTION (provided by applicant): Radiation therapy is one of the mainstays of cancer management. Indeed common clinical practice integrates this therapeutic modality with surgery and chemotherapy into definitive treatment strategies of advanced cancers. Yet despite intensive application of combined modality therapies, significant numbers of radiotherapy patients treated with curative intent ultimately fail. While reasons for radiotherapy failures vary, abnormal tumor microenvironments, tumor progression, and metastatic spread of neoplastic cells are believed to be major contributors. Since these resistance factors are affected by a tumor's ability to develop and maintain a functional blood vessel network, the application of novel vascular targeting approaches in a radiotherapy setting is likely to improve treatment outcomes. Indeed combining strategies that inhibit tumor angiogenesis with radiotherapy can amplify the antitumor effects of radiation. Still, many questions regarding the successful application of this new approach to cancer treatment remain. The central goal of the present application is to develop new insights into the underlying mechanisms of angiosuppressive therapy and to explore avenues to maximize its therapeutic potential. One of the issues to be addressed in this research program is whether the extent of a tumor's inherent vascularity predicates its response to antiangiogenic therapies, i.e. will highly vascular tumors be most susceptible to such interventions? Secondly four color flow cytometric analysis and a green fluorescent protein (GFP) bone marrow transplant model will utilized to investigate the role of circulating endothelial progenitor (CEP) cells in tumor angiogenesis and response to angiosuppressive therapy. Treatments to be examined include those directed at specific aspects of the vascular endothelial growth factor (VEGF) signaling cascade (ligand and VEGF tyrosine kinase inhibition) as well as modulation of the endogenous inhibitor of angiogenesis, endostatin. The former will examine small molecule targeting strategies while the latter will utilize a self-complimentary recombinant adeno associated virus (SC AAV) transduction of skeletal muscle as a platform for angio-suppressive protein delivery. Finally the hypothesis that simultaneously interfering with multiple aspects of angiogenesis will lead to superior responses in tumors will be explored by combining therapies targeting different points in the same signaling pathway or different components of the angiogenic process in general. The ability of the most efficacious vessel targeting strategy will then be tested in a fractionated radiotherapy setting to test its potential to improve treatment outcomes. The central goal of these studies is to examine the potential of applying vascular targeting strategies to enhance the response of solid tumors to radiation therapy. Experiments are designed to investigate the mechanisms underlying the interaction between such therapies and to develop approaches that would maximize the anti-tumor efficacy of such combined treatments.

DESCRIPTION (provided by applicant): Metastasis remains the major cause of therapeutic failure, poor prognosis and high mortality in breast and prostate cancer patients treated with surgery or radiotherapy. Novel approaches to reduce metastatic incidences and improve overall survival of cancer patients clearly are needed. The current proposal is focused on therapeutically targeting a key player in the metastatic cascade; the cysteine protease cathepsin L (CTSL). CTSL is over-expressed and hyper-activated in primary tumors and metastatic lesions in many cancer types, including breast and prostate cancer. Its over-activation correlates with tumor invasiveness and tumor grade, yielding a poor patient prognosis. CTSL initiates migratory and invasive processes through direct degradation of E-Cadherin, extracellular matrix, and basement membrane components. Cathepsins also critically contribute to the process by which metastatic tumor cells in the skeleton stimulate osteoclast-mediated bone resorption; a source of significant morbidity in prostate and breast cancer patients. CTSL may therefore provide a unique target in the metastatic process. The goal of the present proposal is to explore the anti-metastatic potential of CTSL inhibition using a small molecule targeting approach in human (MBA-MB-231, PC-3ML) and rodent (TRAMP) breast and prostate cancer models. Aim 1 will assess the impact of selective small molecule CTSL inhibitors on key functional steps associated with tumor cell dissemination including ECM degradation, cell motility, and cell invasion. In Aim 2 the effect of hypoxic and acidic tumor microenvironments, as commonly occur in solid tumors, on CTSL release, activity, and response to small molecule inhibition will be assessed. Aim 3 will examine whether cathepsin targeting can inhibit bone resorption; a key process leading to skeletal morbidity in cancer patients. Aim 4 will evaluate the in vivo anti-metastatic potential of CTSL targeting using both molecular suppression and selective therapeutic inhibition of CTSL. First, a known number of MDA-MB- 231 or PC-3ML cells will be injected via the intracardiac route and the efficacy of CTSL inhibitors (KGP94 or KGP207) will be determined by (i) non-invasive monitoring of the metastatic burden using an in vivo imaging system, and (ii) quantifying the number of bone metastases. Second, select studies will evaluate the effect of CTSL interference on the spread of cancer cells from primary tumors to secondary sites in (i) mice undergoing surgical removal of orthotopically transplanted breast tumors and (ii) the spontaneous prostate cancer metastasis TRAMP model. Finally, Aim 5 will address the role of CTSL and its inhibition to tumor induced angiogenesis and vasculogenesis, key contributors to metastasis. The successful completion of the proposed studies will provide evidence that CTSL is a potential therapeutic target for novel anti-metastatic cancer therapies. Preclinical investigations of approaches targeting the spread of cancer cells may ultimately provide clinical benefit by enabling the development of future treatment regimes designed to enhance outcomes of existing surgical interventions and radiotherapy treatments for advanced breast and prostate cancer patients.

? DESCRIPTION (provided by applicant): Prostate cancer is the most commonly diagnosed cancer in North America and the leading cause of cancer death in men. Metastasis is the primary cause of therapeutic failure in prostate cancer patients; resulting in poor prognosis and high mortality. Hypoxia, a common feature of most solid tumors including prostate cancer, has long been associated with enhanced metastasis and poor patient outcome. Hypoxia arises in tumors because the aggressive growth of neoplastic cells and associated over-expression of pro-angiogenic factors leads to aberrant vascular networks that are incapable of adequately delivering nutrients and removing waste products. As a consequence tumor cells can experience oxygen deprivation at the limit of oxygen diffusion (chronic or diffusion-limited hypoxia) or due to intermittent blood flow fluctuations (acute or perfusion-limited hypoxia). Both have been linked to enhanced tumor cell spread though their relative importance has not been unequivocally resolved. Evidence indicates that oxygen deficiencies in tumors can directly impact critical tumor cell functions critical to the metastatic phenotype including proliferation, survival, invasion, and induction of angiogenesis. The key hypoxia-regulated transcriptional factor HIF1 drives expression of many of the genes associated with these cancer cell functions. The Src family kinases (SFKs) are a family of non-receptor tyrosine kinases that are frequently hyper-activated in human cancers. In prostate cancer they contribute to increased aggressiveness and are associated with hormone insensitivity and metastasis. Src signaling promotes not only an invasive tumor cell phenotype but also enhances angiogenesis and formation of bone metastases. Interestingly, our preliminary evidence indicates that hypoxia is able to promote Src signaling, a less well-recognized finding. The goals of this proposal are to (i) determine the relative impact of acute and chronic hypoxic exposures on SFK activity and metastatic potential of prostate cancer cells in vitro and in vivo, (ii) examine the hypoxia-mediated HIF1-Src signaling pathways, and (iii) assess the ability of clinically employed small molecule Src inhibitors to impair hypoxia-induced metastasis. We believe that treatment options such as Src targeting strategies that interfere with the process of metastasis will have significan impact on cancer therapy outcomes and related mortality. Since metastasis has been linked both clinically and experimentally to hypoxia gaining a better understanding of how the varying oxygen deprivations that can occur in solid tumors impact metastasis-associated signaling and behavior should ultimately lead to the development of novel treatment options in the future.