Summary: A theoretical model of blood -brain exchange is developed and a procedure is derived that can be used for graphing multiple-time tissue uptake data and deter mining whether a unidirectional transfer process was

Adoptive transfer of large numbers of tumor-reactive CD8+ cytotoxic T lymphocytes (CTLs) expanded and differentiated in vitro has shown promising clinical activity against cancer. However, such protocols are complicated by extensive ex vivo manipulations of tumor-reactive cells and have largely focused on CD8+ CTLs, with much less emphasis on the role and contribution of CD4+ T cells. Using a mouse model of advanced melanoma, we found that transfer of small numbers of naive tumor-reactive CD4+ T cells into lymphopenic recipients induces substantial T cell expansion, differentiation, and regression of large established tumors without the need for in vitro manipulation. Surprisingly, CD4+ T cells developed cytotoxic activity, and tumor rejection was dependent on class II–restricted recognition of tumors by tumor-reactive CD4+ T cells. Furthermore, blockade of the coinhibitory receptor CTL-associated antigen 4 (CTLA-4) on the transferred CD4+ T cells resulted in greater expansion of effector T cells, diminished accumulation of tumor-reactive regulatory T cells, and superior antitumor activity capable of inducing regression of spontaneous mouse melanoma. These findings suggest a novel potential therapeutic role for cytotoxic CD4+ T cells and CTLA-4 blockade in cancer immunotherapy, and demonstrate the potential advantages of differentiating tumor-reactive CD4+ cells in vivo over current protocols favoring in vitro expansion and differentiation.

Summary:The method of graphical analysis for the eval uation of sequential data (e.g., tissue and blood concen trations over time) in which the test substance is irre versibly trapped in the system has been expanded. A sim pler derivation of the original analysis is presented.General equations are derived that can be used to analyze tissue uptake data when the blood-plasma concentration of the test substance cannot be easily measured. In ad dition, general equations are derived for situations when trapping of the test substance is incomplete and for a combination of these two conditions. These derivationsThe study of the movement of solute molecules across tissue capillaries and their localization within selected tissues of both animal and human subjects has been greatly facilitated by the use of tomo graphic machines. These instruments can provide sequential measurements over time of regional tissue concentrations. The general experimental protocol usually involves the injection of the test substance into the blood of the subject and then measuring the concentration of the substance in both the blood and various tissue regions over a period of time. These data may then be analyzed by a number of methods to ascertain the desired parameters of the system.One method of analysis that has been proposed for those substances that are irreversibly trapped in the system is a graphical analysis of multiple time data points . For this method, an equation has been developed for a very general model. When certain measurable quantities are This approach is also shown to result in equations with at least one less nonlinear term than those derived from direct compartmental analysis. Specific applications of these equations are illustrated for a compartmental system with one reversible region (with or without re versible binding) and one irreversible region.

TGF-␤ can signal by means of Smad transcription factors, which are quintessential tumor suppressors that inhibit cell proliferation, and by means of Smad-independent mechanisms, which have been implicated in tumor progression. Although Smad mutations disable this tumor-suppressive pathway in certain cancers, breast cancer cells frequently evade the cytostatic action of TGF-␤ while retaining Smad function. Through immunohistochemical analysis of human breast cancer bone metastases and functional imaging of the Smad pathway in a mouse xenograft model, we provide evidence for active Smad signaling in human and mouse bonemetastatic lesions. Genetic depletion experiments further demonstrate that Smad4 contributes to the formation of osteolytic bone metastases and is essential for the induction of IL-11, a gene implicated in bone metastasis in this mouse model system. Activator protein-1 is a key participant in Smad-dependent transcriptional activation of IL-11 and its overexpression in bone-metastatic cells. Our findings provide functional evidence for a switch of the Smad pathway, from tumor-suppressor to prometastatic, in the development of breast cancer bone metastasis.IL-11 ͉ Smad4 ͉ TGF-␤ T GF-␤ plays a crucial role as a growth-inhibitory cytokine in many tissues (1, 2). The cytostatic effect of TGF-␤ is mediated by a serine͞threonine kinase receptor complex that phosphorylates Smad2 and Smad3, which then translocate into the nucleus and bind Smad4 to generate transcriptional regulatory complexes (3). SMAD4 (also known as Deleted in Pancreatic Carcinoma locus 4 or DPC4) and, to a lesser extent, SMAD2 suffer mutational inactivation in a proportion of pancreatic and colon cancers (1, 2). However, tumor cells that evade this antiproliferative control by other mechanisms may display an altered sensitivity to TGF-␤ and undergo tumorigenic progression in response to this cytokine (1, 2). Patients whose pancreatic or colon tumors express TGF-␤ receptors fare less well than those with low or absent TGF-␤ receptor expression in the tumor (4). In mouse models of breast cancer, TGF-␤ signaling promotes lung (5, 6) and bone metastasis (7). In the case of osteolytic bone metastasis by breast cancer cells, it has been proposed that TGF-␤ released from the decaying bone matrix stimulates neighboring tumor cells, establishing a vicious cycle that exacerbates the growth of the metastatic lesion (8).The TGF-␤ signaling mechanisms that foster metastasis in human cancer are an important open question and a subject of debate. Because Smad factors are quintessential tumor suppressors, the basis for the protumorigenic effects of TGF-␤ has been sought in the Smad-independent signaling pathways that may be triggered by TGF-␤. Results obtained by means of overexpression of dominant negative mutant components of the Rho pathway (9, 10) or pharmacologic inhibitors of p38 mitogen-activated protein kinase (11, 12) have implicated these pathways in the proinvasive and metastatic effects of TGF-␤ in transformed cells. In contrast, results obta...

We used bioluminescence imaging to reveal patterns of metastasis formation by human breast cancer cells in immunodeficient mice. Individual cells from a population established in culture from the pleural effusion of a breast cancer patient showed distinct patterns of organ-specific metastasis. Single-cell progenies derived from this population exhibited markedly different abilities to metastasize to the bone, lung, or adrenal medulla, which suggests that metastases to different organs have different requirements. Transcriptomic profiling revealed that these different single-cell progenies similarly express a previously described "poor-prognosis" gene expression signature. Unsupervised classification using the transcriptomic data set supported the hypothesis that organ-specific metastasis by breast cancer cells is controlled by metastasis-specific genes that are separate from a general poor-prognosis gene expression signature. Furthermore, by using a gene expression signature associated with the ability of these cells to metastasize to bone, we were able to distinguish primary breast carcinomas that preferentially metastasized to bone from those that preferentially metastasized elsewhere. These results suggest that the bone-specific metastatic phenotypes and gene expression signature identified in a mouse model may be clinically relevant.

Summary:Positron emission tomography studies with the opiate antagonist [18F]cyclofoxy ([18F]CF) were per formed in baboons. Bolus injection studies demonstrated initial uptake dependent on blood flow. The late uptake showed highest binding in caudate nuclei, amygdala, thal amus, and brainstem and the least accumulation in cere bellum. By 60 min postinjection, regional brain radioac tivity cleared at the same rate as metabolite-corrected plasma, i.e., transient equilibrium was achieved. Com partmental modeling methods were applied to time activity curves from brain and metabolite-corrected plasma. Individual rate constants were estimated with poor precision. The model estimate of the total volume of distribution (VT), representing the ratio of tissue radioac tivity to metabolite-corrected plasma at equilibrium, was reliably determined. The apparent volume of distribution Va significantly overestimated V T and produced artifi cially high image contrast. These differences were pre dicted by compartment model theory and were caused by a plasma clearance rate that was close to the slowest tissue clearance rate. To develop a simple method to mea sure V T' an infusion protocol consisting of bolus plus continuous infusion (B/I) of CF was designed and applied in a separate set of studies. The Va values from the B/I studies agreed with the V T values from both B/I and bolus studies. This infusion approach can produce accurate re ceptor measurements and has the potential to shorten scan time and simplify the acquisition and processing of scan and blood data.

The inability to visualize the true extent of cancers represents a significant challenge in many areas of oncology. The margins of most cancer types are not well demarcated because the cancer diffusely infiltrates the surrounding tissues. Furthermore, cancers may be multifocal and characterized by the presence of microscopic satellite lesions. Such microscopic foci represent a major reason for persistence of cancer, local recurrences, and metastatic spread and are usually impossible to visualize with currently available imaging technologies. An imaging method to reveal the tumor extent is desired clinically and surgically. Here we show the precise visualization of tumor margins, microscopic tumor invasion, and multifocal loco-regional tumor spread using a new generation of surface-enhanced resonance Raman scattering (SERRS) nanoparticles, which are termed here SERRS-nanostars. The SERRS-nanostars feature a star-shaped gold core, a Raman reporter resonant in the near-infrared spectrum, and a primer-free silication method. In mouse models of pancreatic cancer, breast cancer, prostate cancer, and sarcoma, SERRS-nanostars enabled accurate detection of macroscopic malignant lesions as well as microscopic disease, without the need for a targeting moiety. Moreover, the sensitivity (1.5 femtomolar limit of detection under in vivo Raman imaging conditions) of SERRS-nanostars allowed imaging of premalignant lesions of pancreatic and prostatic neoplasias. High sensitivity and broad applicability, in conjunction with their inert gold-silica composition, render SERRS-nanostars a promising imaging agent for more precise cancer imaging and resection.

We used bioluminescence imaging to reveal patterns of metastasis formation by human breast cancer cells in immunodeficient mice. Individual cells from a population established in culture from the pleural effusion of a breast cancer patient showed distinct patterns of organ-specific metastasis. Single-cell progenies derived from this population exhibited markedly different abilities to metastasize to the bone, lung, or adrenal medulla, which suggests that metastases to different organs have different requirements. Transcriptomic profiling revealed that these different single-cell progenies similarly express a previously described "poor-prognosis" gene expression signature. Unsupervised classification using the transcriptomic data set supported the hypothesis that organ-specific metastasis by breast cancer cells is controlled by metastasis-specific genes that are separate from a general poor-prognosis gene expression signature. Furthermore, by using a gene expression signature associated with the ability of these cells to metastasize to bone, we were able to distinguish primary breast carcinomas that preferentially metastasized to bone from those that preferentially metastasized elsewhere. These results suggest that the bone-specific metastatic phenotypes and gene expression signature identified in a mouse model may be clinically relevant.