Global Dormancy of Metastases Due to Systemic Inhibition of Angiogenesis

Autopsy studies of adults dying of non-cancer causes have shown that virtually all of us possess occult, cancerous lesions. This suggests that, for most individuals, cancer will become dormant and not progress, while only in some will it become symptomatic disease. Meanwhile, it was recently shown in animal models that a tumor can produce both stimulators and inhibitors of its own blood supply. To explain the autopsy findings in light of the preclinical research data, we propose a mathematical model of cancer development at the organism scale describing a growing population of metastases, which, together with the primary tumor, can exert a progressively greater level of systemic angiogenesis-inhibitory influence that eventually overcomes local angiogenesis stimulation to suppress the growth of all lesions. As a departure from modeling efforts to date, we look not just at signaling from and effects on the primary tumor, but integrate over this increasingly negative global signaling from all sources to track the development of total tumor burden. This in silico study of the dynamics of the tumor/metastasis system identifies ranges of parameter values where mutual angio-inhibitory interactions within a population of tumor lesions could yield global dormancy, i.e., an organism-level homeostatic steady state in total tumor burden. Given that mortality arises most often from metastatic disease rather than growth of the primary per se, this finding may have important therapeutic implications.     

Patch quality and context, but not patch number, drive multi-scale colonization dynamics in experimental aquatic landscapes

Colonization and extinction are primary drivers of local population dynamics, community structure, and spatial patterns of biological diversity. Existing paradigms of island biogeography, metapopulation biology, and metacommunity ecology, as well as habitat management and conservation biology based on those paradigms, emphasize patch size, number, and isolation as primary characteristics influencing colonization and extinction. Habitat selection theory suggests that patch quality could rival size, number, and isolation in determining rates of colonization and resulting community structure. We used naturally colonized experimental landscapes to address four issues: (a) how do colonizing aquatic beetles respond to variation in patch number, (b) how do they respond to variation in patch quality, (c) does patch context affect colonization dynamics, and (d) at what spatial scales do beetles respond to habitat variation? Increasing patch number had no effect on per patch colonization rates, while patch quality and context were critical in determining colonization rates and resulting patterns of abundance and species richness at multiple spatial scales. We graphically illustrate how variation in immigration rates driven by perceived predation risk (habitat quality) can further modify dynamics of the equilibrium theory of island biogeography beyond predator-driven effects on extinction rates. Our data support the importance of patch quality and context as primary determinants of colonization rate, occupancy, abundance, and resulting patterns of species richness, and reinforce the idea that management of metapopulations for species preservation, and metacommunities for local and regional diversity, should incorporate habitat quality into the predictive equation.     

Estradiol receptor levels in human breast carcinomas.

The presence or absence of a specific estradiol-binding protein receptor in the cytoplasm of primary and secondary tumour cells has been used by physicians as an important guide in deciding whether to use hormonal therapy for patients with metastatic breast cancer. This report gives the levels of estradiol receptors in the cytosol of 228 primary and secondary breast tumours, measured by a sensitive multiple-point assay in which dextran-coated charcoal separated bound form unbound estrogen. The data were analysed with a Scatchard plot. Of the 175 primary and 53 secondary tumours 53% and 32% respectively gave positive results. The mean receptor level in the primary tumours was significantly higher among older patients and increased with age. With metastatic lesions positive results were more common in lymph node samples tha in skin nodule samples.     

Spatial dynamics of invasion: the geometry of introduced species

Many exotic species combine low probability of establishment at each introduction with rapid population growth once introduction does succeed. To analyse this phenomenon, we note that invaders often cluster spatially when rare, and consequently an introduced exotic's population dynamics should depend on locally structured interactions. Ecological theory for spatially structured invasion relies on deterministic approximations, and determinism does not address the observed uncertainty of the exotic-introduction process. We take a new approach to the population dynamics of invasion and, by extension, to the general question of invasibility in any spatial ecology. We apply the physical theory for nucleation of spatial systems to a lattice-based model of competition between plant species, a resident and an invader, and the analysis reaches conclusions that differ qualitatively from the standard ecological theories. Nucleation theory distinguishes between dynamics of single- and multi-cluster invasion. Low introduction rates and small system size produce single-cluster dynamics, where success or failure of introduction is inherently stochastic. Single-cluster invasion occurs only if the cluster reaches a critical size, typically preceded by a number of failed attempts. For this case, we identify the functional form of the probability distribution of time elapsing until invasion succeeds. Although multi-cluster invasion for sufficiently large systems exhibits spatial averaging and almost-deterministic dynamics of the global densities, an analytical approximation from nucleation theory, known as Avrami's law, describes our simulation results far better than standard ecological approximations.     

A dynamical model for the growth and size distribution of multiple metastatic tumors

Metastasis is the spread of tumors culminating in the establishment of one or more secondary tumors at remote sites. In deciding the best treatment for cancer therapy, estimations of the colony size of metastatic tumors and predictions of the future spread of colonies are needed. A dynamical model for the colony size distribution of multiple metastatic tumors is presented here. The dynamics is described by equations that incorporate both the colonization by metastasis and the growth of each colony. When the colony growth is subject to the Gompertz function, the explicit solution obtained tends to an asymptotic stable distribution that shows a monotonically decreasing or U-shaped pattern according to the values of clinically significant parameters, such as the colonization coefficient and the fractal dimension of blood vessels. This predicted colony size distribution agrees well with successive data of a clinically observed size distribution of multiple metastatic tumors of liver cancer. The combined analysis of the theoretical colony size distribution and clinical data will give useful information on the diagnosis and the therapy for cancer patients.     

Secreted Gaussia Luciferase as a Biomarker for Monitoring Tumor Progression and Treatment Response of Systemic Metastases

Background

Currently, only few techniques are available for quantifying systemic metastases in preclinical model. Thus techniques that can sensitively detect metastatic colonization and assess treatment response in real-time are urgently needed. To this end, we engineered tumor cells to express a naturally secreted Gaussia luciferase (Gluc), and investigated its use as a circulating biomarker for monitoring viable metastatic or primary tumor growth and their treatment responses.

Methodology/Principal Findings

We first developed orthotopic primary and metastatic breast tumors with derivative of MDA-MB-231 cells expressing Gluc. We then correlated tumor burden with Gluc activity in the blood and urine along with bioluminescent imaging (BLI). Second, we utilized blood Gluc assay to monitor treatment response to lapatinib in an experimental model of systemic metastasis. We observed good correlation between the primary tumor volume and Gluc concentration in blood (R² = 0.84) and urine (R² = 0.55) in the breast tumor model. The correlation deviated as a primary tumor grew due to a reduction in viable tumor fraction. This was also supported by our mathematical models for tumor growth to compare the total and viable tumor burden in our model. In the experimental metastasis model, we found numerous brain metastases as well as systemic metastases including bone and lungs. Importantly, blood Gluc assay revealed early growth of metastatic tumors before BLI could visualize their presence. Using secreted Gluc, we localized systemic metastases by BLI and quantitatively monitored the total viable metastatic tumor burden by blood Gluc assay during the course of treatment with lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER2.

Conclusion/Significance

We demonstrated secreted Gluc assay accurately reflects the amount of viable cancer cells in primary and metastatic tumors. Blood Gluc activity not only tracks metastatic tumor progression but also serves as a longitudinal biomarker for tumor response to treatments.     

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.     

Use of tumour marker immunoreactivity to identify primary site of metastatic cancer.

OBJECTIVES--To determine whether variations in the expression of tumour related antigens can predict the origin of tumours. DESIGN--Immunohistological study of tumour marker expression in primary adenocarcinomas and respective metastatic deposits. Antibodies to the following tumour markers were used: polymorphic epithelial mucin (NCRC-11 and SM3), carcinoembryonic antigen, carcinoembryonic antigen with non-specific antigen co-specificity, CA125, CA19.9, prostate specific antigens, and thyroglobulin. SETTING--Histopathology department of teaching hospital. SUBJECTS--100 pathology sections of metastatic adenocarcinoma and their related primary tumours. MAIN OUTCOME MEASURES--Concordance of reactivity between primary and metastatic tumours. Reactivity profiles of tumour sites. RESULTS--The correct primary site of origin was predicted in 70% (33/47) of tumours in men and 54% (27/43) tumours in women with antibodies SM3, 288, CA19.9, CA125, and PSA (men only). Specificities ranged from 68% for breast tumour to 98% for prostate tumour. CONCLUSION--Use of tumour markers in patients presenting with metastatic adenocarcinoma of unknown origin can help localise the probable primary sites and reduce the need for extensive and expensive imaging techniques.     

Anti-metastatic effects of viral and non-viral mediated Nk4 delivery to tumours

The most common cause of death of cancer sufferers is through the occurrence of metastases. The metastatic behaviour of tumour cells is regulated by extracellular growth factors such as hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, and aberrant expression/activation of the c-Met receptor is closely associated with metastatic progression. Nk4 (also known as Interleukin (IL)32b) is a competitive antagonist of the HGF c-Met system and inhibits c-Met signalling and tumour metastasis. Nk4 has an additional anti-angiogenic activity independent of its HGF-antagonist function. Angiogenesis-inhibitory as well as cancer-specific apoptosis inducing effects make the Nk4 sequence an attractive candidate for gene therapy of cancer. This study investigates the inhibition of tumour metasasis by gene therapy mediated production of Nk4 by the primary tumour. Optimal delivery of anti-cancer genes is vital in order to achieve the highest therapeutic responses. Non-viral plasmid delivery methods have the advantage of safety and ease of production, providing immediate transgene expression, albeit short-lived in most tumours. Sustained presence of anti-angiogenic molecules is preferable with anti-angiogenic therapies, and the long-term expression mediated by Adeno-associated Virus (AAV) might represent a more appropriate delivery in this respect. However, the incubation time required by AAV vectors to reach appropriate gene expression levels hampers efficacy in many fast-growing murine tumour models. Here, we describe murine trials assessing the effects of Nk4 on the spontaneously metastatic Lewis Lung Carcinoma (LLC) model when delivered to primary tumour via plasmid lipofection or AAV2 vector. Intratumoural AAV-Nk4 administration produced the highest therapeutic response with significant reduction in both primary tumour growth and incidence of lung metastases. Plasmid-mediated therapy also significantly reduced metastatic growth, but with moderate reduction in primary subcutaneous tumour growth. Overall, this study demonstrates the potential for Nk4 gene therapy of metastatic tumours, when delivered by AAV or non-viral methods.     

Cytological and immunocytochemical evaluation of thyroid and breast masses in patients with a previous neoplasm: case reports

The diagnosis of secondary tumours represents one of the most important fields in the application of fine needle aspiration cytology (FNAC). We studied two patients, one with a history of breast cancer and one with a previous tumour of the thyroid, who showed a second mass, in the thyroid and in the breast, respectively, during follow up. The aim of our study was to evaluate if cytology, performed on FNAC smears, may distinguish a metastatic lesion from a second primary tumour, or if further immunocytochemistry should be performed. Our data demonstrate that, while cytology may be indicative of a second primary tumour, the histotype should be confirmed by immunocytochemical staining.     

A mathematical model of the impact of infused targeted cytotoxic agents on brain tumours: implications for detection,design and delivery

Abstract. Motivated by the recent development of highly specific agents for brain tumours, we develop a mathematical model of the spatio-temporal dynamics of a brain tumour that receives an infusion of a highly specific cytotoxic agent (e.g. IL-4-PE, a cytotoxin comprised of IL-4 and a mutated form of Pseudomonas exotoxin). We derive an approximate but accurate mathematical formula for the tumour cure probability in terms of the tumour characteristics (size at time of detection, proliferation rate, diffusion coefficient), drug design (killing rate, loss rate and convection constants for tumour and tissue), and drug delivery (infusion rate, infusion duration). Our results suggest that high specificity is necessary but not sufficient to cure malignant gliomas; a nondispersed spatial profile of pretreatment tumour cells and/or good drug penetration are also required. The most important levers to improve tumour cure appear to be earlier detection, higher infusion rate, lower drug clearance rate and better convection into tumour, but not tissue. In contrast, the tumour cure probability is less sensitive to a longer infusion duration and enhancements in drug potency and drug specificity.     

Proliferative activity of primary breast cancer and of synchronous lymph node metastases evaluated by [3H]-thymidine labelling index

Abstract. The [³H]-thymidine labelling index ([³H]TdR LI) has been used to evaluate and comparatively analyse the proliferative activity of different tumour lesions from the same patient. The analysis was performed on the primary tumour and its synchronous lymph node metastasis from 210 patients operated on for breast cancer. A direct relation was observed between the proliferative activity of the two different lesions (Spearman correlation coefficient = 0–46, P< 00001), but there was considerable scatter amongst the data. The [³H]TdR LI of primary and of metastatic lesions belonged to the same proliferation classes in only 47% of the cases. Higher or lower [³H]TdR LI values, categorized on the basis of the tertiles of the frequency distribution, occurred in the node metastasis than in the primary tumour in an almost similar percentage of the remaining cases. Menopause, receptor status and pathological features did not affect interlesion kinetic patterns. The prognostic role of the proliferative activity of the two different lesions was investigated on 107 patients with stage II tumours homogeneously treated with surgery and systemic adjuvant therapy. Relapse-free survival at 3 years was significantly affected by the proliferative activity of the primary tumour but not by that of the lymph node metastasis.     

Proteomic evaluation of sheep serum proteins

Background

Mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. Chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. In particular, the chemokine/receptor pair SDF-1/CXCR4 has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. The aim of this study was the immunohistochemical defininition of the expression profile of CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation.

Results

A total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas), 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. Tissues were processed for standard histological examination, and all lesions were classified according to the World Health Organization criteria. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 score evaluating both the number of positive cells and the intensity of staining. Six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did not show CXCR4 immunoreactivity. CXCR4 score was statistically significantly associated with the histological features of the samples, showing an increase accordingly with the degree of neoplastic transformation (from normal tissue to metastatic lesions). Finally, in the primary cultures obtained from 6 primary feline mammary carcinomas CXCR4 expression was detected in all cells and its activation by SDF-1 in vitro treatment caused a significant increase in the proliferation rate in 5 out of 6 tumours.

Conclusions

These results indicate that malignant feline mammary tumours commonly express CXCR4, with a higher level in malignant tumours, and, in most of the cases analysed, metastatic cells display stronger immunoreactivity for CXCR4 than the corresponding primary tumours. Moreover, CXCR4 activation in primary cultures of feline mammary carcinomas causes increase in the proliferative rate. Thus, SDF-1/CXCR4 system seems to play a tumorigenic in feline mammary gland malignancy and in vitro cultures from these tumour samples may represent an experimental model to investigate the biological and pharmacological role of this chemokinergic axis.     

Tumour-stromal interactions in acid-mediated invasion: A mathematical model

It is well established that the tumour microenvironment can both promote and suppress tumour growth and invasion, however, most mathematical models of invasion view the normal tissue as inhibiting tumour progression via immune modulation or spatial constraint. In particular, the production of acid by tumour cells and the subsequent creation of a low extracellular pH environment has been explored in several ‘acid-mediated tumour invasion’ models where the acidic environment facilitates normal cell death and permits tumour invasion. In this paper, we extend the acid-invasion model developed by Gatenby and Gawlinski (1996) to include both the competitive and cooperative interactions between tumour and normal cells, by incorporating the influence of extracellular matrix and protease production at the tumour-stroma interface. Our model predicts an optimal level of tumour acidity which produces both cell death and matrix degradation. Additionally, very aggressive tumours prevent protease production and matrix degradation by excessive normal cell destruction, leading to an acellular (but matrix filled) gap between the tumour and normal tissue, a feature seen in encapsulated tumours. These results sugest, counterintuitively, that increasing tumour acidity may, in some cases, prevent tumour invasion.     

Differential Expression of Prognostic Proteomic Markers in Primary Tumour,Venous Tumour Thrombus and Metastatic Renal Cell Cancer Tissue and Correlation with Patient Outcome

Renal cell carcinoma (RCC) is the most deadly of urological malignancies. Metastatic disease affects one third of patients at diagnosis with a further third developing metastatic disease after extirpative surgery. Heterogeneity in the clinical course ensures predicting metastasis is notoriously difficult, despite the routine use of prognostic clinico-pathological parameters in risk stratification. With greater understanding of pathways involved in disease pathogenesis, a number of biomarkers have been shown to have prognostic significance, including Ki67, p53, vascular endothelial growth factor receptor 1 (VEGFR1) and ligand D (VEGFD), SNAIL and SLUG. Previous pathway analysis has been from study of the primary tumour, with little attention to the metastatic tumours which are the focus of targeted molecular therapies. As such, in this study a tissue microarray from 177 patients with primary renal tumour, renal vein tumour thrombus and/or RCC metastasis has been created and used with Automated Quantitative Analysis (AQUA) of immunofluorescence to study the prognostic significance of these markers in locally advanced and metastatic disease. Furthermore, this has allowed assessment of differential protein expression between the primary tumours, renal vein tumour thrombi and metastases. The results demonstrate that clinico-pathological parameters remain the most significant predictors of cancer specific survival; however, high VEGFR1 or VEGFD can predict poor cancer specific survival on univariate analysis for locally advanced and metastatic disease. There was significantly greater expression of Ki67, p53, VEGFR1, SLUG and SNAIL in the metastases compared with the primary tumours and renal vein tumour thrombi. With the exception of p53, these differences in protein expression have not been shown previously in RCC. This confirms the importance of proliferation, angiogenesis and epithelial to mesenchymal transition in the pathogenesis and metastasis of RCC. Importantly, this work highlights the need for further pathway analysis of metastatic tumours for overcoming drug resistance and developing new therapies.     

The biological consequences of altered MHC class I expression in tumours.

The identification of different mechanisms by which tumours escape from the immune system has helped to evaluate the clinical relevance of a variety of phenotypic changes that occur during tumour development. Among them, changes in HLA class I expression play a leading role in the tumour-host environment since HLA class I molecules interact with T lymphocytes for antigen presentation and with NK cells for inhibition/activation of these immune effector cells. Our laboratory has proposed a classification of the altered HLA class I phenotypes frequently found in human tumours, into five major groups. This review focuses on the tumour phenotypes found in primary and metastatic lesions, the molecular mechanisms that give rise to each phenotype and the clinical implications of these findings.