Less is more: reducing the number of administered chimeric antigen receptor T cells in a mouse model using a mathematically guided approach.

Chimeric antigen receptor T cell (CAR-T) therapy is a novel approved treatment for hematological malignancies, still under development for solid tumors. Here, we use a rate equation-based mathematical model to discover regimens and schedules that maintain efficacy while potentially reducing toxicity by decreasing the amount of CAR-T infused. Tested on an in vivo murine model of spontaneous breast cancer, we show that our mathematical model accurately recapitulates in vivo tumor growth results achieved in the previous experiments. Moreover, we use the mathematical model to predict results of new therapy schedules and successfully prospectively validated these predictions in the in vivo. We conclude that using one tenth and even one percent of a full CAR-T dose used in preclinical trials can achieve efficacious results similar to full dose treatment.

Nanoparticulate vaccine inhibits tumor growth via improved T cell recruitment into melanoma and huHER2 breast cancer.

Nanoparticulate vaccines are promising tools to overcome cancer immune evasion. However, a deeper understanding on nanoparticle-immune cell interactions and treatments regime is required for optimal efficacy. We provide a comprehensive study of treatment schedules and mode of antigen-association to nanovaccines on the modulation of T cell immunity in vivo, under steady-state and tumor-bearing mice. The coordinated delivery of antigen and two adjuvants (Monophosphoryl lipid A, oligodeoxynucleotide cytosine-phosphate-guanine motifs (CpG)) by nanoparticles was crucial for dendritic cell activation. A single vaccination dictated a 3-fold increase on cytotoxic memory-T cells and raised antigen-specific immune responses against B16.M05 melanoma. It generated at least a 5-fold increase on IFN-γ cytokine production, and presented over 50% higher lymphocyte count in the tumor microenvironment, compared to the control. The number of lymphocytes at the tumor site doubled with triple immunization. This lymphocyte infiltration pattern was confirmed in mammary huHER2 carcinoma, with significant tumor reduction.

Elimination of progressive mammary cancer by repeated administrations of chimeric antigen receptor-modified T cells.

Continuous oncogenic processes that generate cancer require an on-going treatment approach to eliminate the transformed cells, and prevent their further development. Here, we studied the ability of T cells expressing a chimeric antibody-based receptor (CAR) to offer a therapeutic benefit for breast cancer induced by erbB-2. We tested CAR-modified T cells (T-bodies) specific to erbB-2 for their antitumor potential in a mouse model overexpressing a human erbB-2 transgene that develops mammary tumors. Comparing the antitumor reactivity of CAR-modified T cells under various therapeutic settings, either prophylactic, prior to tumor development, or therapeutically. We found that repeated administration of CAR-modified T cells is required to eliminate spontaneously developing mammary cancer. Systemic, as well as intratumoral administered CAR-modified T cells accumulated at tumor sites and eventually eliminated the malignant cells. Interestingly, within a few weeks after a single CAR T cells' administration, and rejection of primary lesion, tumors usually relapsed both in treated mammary gland and at remote sites; however, repeated injections of CAR-modified T cells were able to control the secondary tumors. Since spontaneous tumors can arise repeatedly, especially in the case of syndromes characterized by specific susceptibility to cancer, multiple administrations of CAR-modified T cells can serve to control relapsing disease.

Suppression of murine colitis and its associated cancer by carcinoembryonic antigen-specific regulatory T cells.

The adoptive transfer of regulatory T cells (Tregs) offers a promising strategy to combat pathologies that are characterized by aberrant immune activation, including graft rejection and autoinflammatory diseases. Expression of a chimeric antigen receptor (CAR) gene in Tregs redirects them to the site of autoimmune activity, thereby increasing their suppressive efficiency while avoiding systemic immunosuppression. Since carcinoembryonic antigen (CEA) has been shown to be overexpressed in both human colitis and colorectal cancer, we treated CEA-transgenic mice that were induced to develop colitis with CEA-specific CAR Tregs. Two disease models were employed: T-cell-transfer colitis as well as the azoxymethane-dextran sodium sulfate model for colitis-associated colorectal cancer. Systemically administered CEA-specific (but not control) CAR Tregs accumulated in the colons of diseased mice. In both model systems, CEA-specific CAR Tregs suppressed the severity of colitis compared to control Tregs. Moreover, in the azoxymethane-dextran sodium sulfate model, CEA-specific CAR Tregs significantly decreased the subsequent colorectal tumor burden. Our data demonstrate that CEA-specific CAR Tregs exhibit a promising potential in ameliorating ulcerative colitis and in hindering colorectal cancer development. Collectively, this study provides a proof of concept for the therapeutic potential of CAR Tregs in colitis patients as well as in other autoimmune inflammatory disorders.

Redirected T cells that target pancreatic adenocarcinoma antigens eliminate tumors and metastases in mice.

BACKGROUND & AIMS: Pancreatic adenocarcinoma (PAC) is often diagnosed at an advanced and inoperable stage, and standard systemic treatments are generally ineffective. We investigated the effects of adoptive transfer of tumor-specific T cells that express chimeric antibody-based receptors (CAR) to mice with primary and metastatic PAC xenografts. METHODS: Human effector T cells were genetically modified to express CAR against Her2/neu or CD24, a putative PAC stem cell antigen. The antitumor reactivity of the engineered T cells (T-bodies) was evaluated in SCID mice with different PAC xenografts. A total of 1 × 10(7) T-bodies were injected via the tail vein or directly administered to the subcutaneous tumor on 3 or 4 alternating days. Mice were then given twice-daily intraperitoneal injections of interleukin-2 for 10 days. RESULTS: Intratumor injection of human CD24 and Her2/neu-specific T-bodies completely eliminated the tumors from most animals. Intravenous injection of T-bodies reduced tumor size and prolonged survival of mice with orthotopically transplanted tumors; more than 50% of animals appeared to be disease-free more than 2 months later. Additional systemic administration of T-bodies 8 weeks after the initial injection eliminated primary tumors, along with liver and draining lymph node metastases. A single administration of the Her2/neu-specific T-bodies prolonged the survival of mice with tumors in which most of the cells expressed the target antigen. In contrast, the CD24-specific T-bodies prolonged survival of mice in which only a subpopulation of the tumor cells expressed the antigen. CONCLUSIONS: CAR-redirected T cells stop growth and metastasis of PAC xenografts in mice. T-bodies specific to CD24, a putative cancer stem cell antigen, were effective against PAC xenografts that had only a subset of antigen-expressing cells.

Redirected tumor-specific allogeneic T cells for universal treatment of cancer.

Adoptive cell transfer of allogeneic tumor-specific T cells could potentially be used as a universal treatment for cancer. We present a novel approach for adoptive immunotherapy using fully MHC-mismatched allogeneic T cells redirected with tumor-specific, non-MHC-restricted antibody-based chimeric antigen receptor (T-bodies) in the absence of GVHD. Mice bearing systemic metastatic disease were lymphodepleted by irradiation and treated with Her2/neu re-directed T cells. Lymphodepletion created a 'therapeutic window', which allowed the allo-T-bodies to attack the tumor before their rejection. A single split dose administration of allogeneic T-bodies extended the survival of tumor-bearing mice similarly to syngeneic T-bodies, and to a significantly greater extent than nonspecific allogeneic T cells. Blocking egress of lymphocytes from lymphoid organs using the sphingosine-1-phosphate agonist, FTY720, extended the persistence of allogeneic T cells such that allogeneic T-bodies provided superior therapeutic benefit relative to syngeneic ones, and dramatically extended the median survival time of the treated mice for more than a year. Therefore, we suggest that ex-vivo generated MHC-mismatched T-bodies can be used universally for off-the-shelf cancer immunotherapy and that their graft-versus-host reactivity can be safely harnessed to potentiate adoptive cell therapy.

Mixed alkanethiol monolayers on submicrometric gold patterns: a controlled platform for studying cell-ligand interactions.

Nanoscale organization of surface ligands often has a critical effect on cell-surface interactions. We have developed an experimental system that allows a high degree of control over the 2-D spatial distribution of ligands. As a proof of concept, we used the developed system to study how T-cell activation is independently affected by antigen density and antigen amount per cell. Arrays of submicrometer gold islands at varying surface coverage were defined on silicon by electron beam lithography (EBL). The gold islands were functionalized with alkanethiol self-assembled monolayers (SAMs) containing a small antigen, 2,4,6-trinotrophenyl (TNP), at various densities. Genetically engineered T-cell hybridomas expressing TNP-specific chimeric T-cell antigen receptor (CAR) were cultured on the SAMs, and their activation was assessed by IL-2 secretion and CD69 expression. It was found that, at constant antigen density, activation increased monotonically with the amount of antigen, while at constant antigen amount activation was maximal at an intermediate antigen density, whose value was independent of the amount of antigen.

Nanoscale CAR Organization at the Immune Synapse Correlates with CAR-T Effector Functions.

T cells expressing chimeric antigen receptors (CARs) are at the forefront of clinical treatment of cancers. Still, the nanoscale organization of CARs at the interface of CAR-Ts with target cells, which is essential for TCR-mediated T cell activation, remains poorly understood. Here, we studied the nanoscale organization of CARs targeting CD138 proteoglycans in such fixed and live interfaces, generated optimally for single-molecule localization microscopy. CARs showed significant self-association in nanoclusters that was enhanced in interfaces with on-target cells (SKOV-3, CAG, FaDu) relative to negative cells (OVCAR-3). CARs also segregated more efficiently from the abundant membrane phosphatase CD45 in CAR-T cells forming such interfaces. CAR clustering and segregation from CD45 correlated with the effector functions of Ca++ influx and target cell killing. Our results shed new light on the nanoscale organization of CARs on the surfaces of CAR-Ts engaging on- and off-target cells, and its potential significance for CAR-Ts' efficacy and safety.

Electrically controlled molecular recognition harnessed to activate a cellular response.

Seamless embedment of electronic devices in biological systems is expected to add the outstanding computing power, memory, and speed of electronics to the biochemical toolbox of nature. Such amalgamation requires transduction of electronic signals into biochemical cues that affect cells. Inspired by biology, where pathways are directed by molecular recognition, we propose and demonstrate a generic electrical-to-biological transducer comprising a two-state electronic antigen and a chimeric cell receptor engineered to bind the antigen exclusively in its "on" state. T-cells expressing these receptors remain inactivated with the antigen in its "off" state. Switching the antigen to its "on" state by an electrical signal leads to its recognition by the T-cells and correspondingly to cell activation.

Comparing Intraperitoneal and Intravenous Personalized ErbB2CAR-T for the Treatment of Epithelial Ovarian Cancer.

High-grade serous ovarian carcinoma (HGSOC) is the most common type of epithelial ovarian cancer. The majority of cases are diagnosed at advanced stages, when intraperitoneal (IP) spread has already occurred. Despite significant surgical and chemotherapeutic advances in HGSOC treatment over the past decades, survival rates with HGSOC have only modestly improved. Chimeric antigen receptor (CAR)-T cells enable T cells to directly bind to tumor-associated antigens in a major histocompatibility complex-independent manner, thereby inducing tumor rejection. While CAR-T cell therapy shows great promise in hematological malignancies, its use in solid tumors is limited. Therefore, innovative approaches are needed to increase the specificity of CAR-modified T cells against solid tumors. The aim of this study was to assess the efficacy and safety of intraperitoneal (IP) versus intravenous (IV) CAR-T cell therapy in the treatment of HGSOC. We constructed a CAR that targets the ErbB2/HER2 protein (ErbB2CAR), which is overexpressed in HGSOC, and evaluated the functionality of ErbB2CAR on ovarian cancer cell lines (OVCAR8, SKOV3, and NAR). Our findings show that an IP injection of ErbB2CAR-T cells to tumor-bearing mice led to disease remission and increased survival compared to the IV route. Moreover, we found that IP-injected ErbB2CART cells circulate to a lesser extent, making them safer for non-tumor tissues than IV-injected cells. Further supporting our findings, we show that the effect of ErbB2CAR-T cells on primary HGSOC tumors is correlated with ErbB2 expression. Together, these data demonstrate the advantages of an IP administration of CAR-T cells over IV administration, offering not only a safer strategy but also the potential for counteracting the effect of ErbB2CAR in HGSOC. Significance: IP-injected ErbB2CAR-T cells led to disease remission and increased survival compared to the IV route. These findings demonstrate the advantages of IP administration, offering a safe treatment strategy with the potential for counteracting the effect of ErbB2CAR in HGSOC.

Extracellular Vesicles Derived from Chimeric Antigen Receptor-T Cells: A Potential Therapy for Cancer.

Chimeric antigen receptor (CAR)-T cells are genetically engineered T cells, directed against a tumor-associated antigen. Extracellular vesicles (EVs) derived from CAR-T cells (CAR-T EVs) may preserve CAR-T activity and overcome one of the major obstacles responsible for CAR-T cell failure in patients with solid tumors. This study aimed to compare CAR-T EVs with their parental cells and explore their cell penetration and cytotoxic activity. Anti-HER-2 CARs were stimulated with specific target cells. EVs were isolated from the cell media and characterized for their content and functions. We found that CAR-T EVs contained a mixture of small and large EVs. Stimulated anti-HER-2+ CAR-T EVs expressed lower cytokine levels compared with their parental CAR-T cells (such as interferon gamma). Higher levels of granzyme B were found in CAR-T EVs (≥20 × ) compared with EVs from unstimulated cells (p < 0.001). Anti-HER-2+ CAR-T EVs bound and penetrated specifically into HER-2 expressing target cells. Similar cytotoxic effects measured by caspase-3/7 activity were found in CAR-T cells and their derived EVs. However, while the CAR-T cells induced massive apoptosis during the first 24 h, CAR-T EVs required 60 - 90 h. In summary, CAR-T EVs provide a novel potent immunotherapy approach that may be effective against solid tumors.

P32-specific CAR T cells with dual antitumor and antiangiogenic therapeutic potential in gliomas.

Glioblastoma is considered one of the most aggressive malignancies in adult and pediatric patients. Despite decades of research no curative treatment is available and it thus remains associated with a very dismal prognosis. Although recent pre-clinical and clinical studies have demonstrated the feasibility of chimeric antigen receptors (CAR) T cell immunotherapeutic approach in glioblastoma, tumor heterogeneity and antigen loss remain among one of the most important challenges to be addressed. In this study, we identify p32/gC1qR/HABP/C1qBP to be specifically expressed on the surface of glioma cells, making it a suitable tumor associated antigen for redirected CAR T cell therapy. We generate p32 CAR T cells and find them to recognize and specifically eliminate p32 expressing glioma cells and tumor derived endothelial cells in vitro and to control tumor growth in orthotopic syngeneic and xenograft mouse models. Thus, p32 CAR T cells may serve as a therapeutic option for glioblastoma patients.

Amelioration of colitis by genetically engineered murine regulatory T cells redirected by antigen-specific chimeric receptor.

BACKGROUND & AIMS: The therapeutic application of regulatory T cells (Tregs) for the treatment of inflammatory diseases is limited by the scarcity of antigen-specific Tregs. A preferred approach to endow effector T cells (Teff) with a desired specificity uses chimeric immune receptors with antibody-type specificity. Accordingly, employing such chimeric immune receptors to redirect Tregs to sites of inflammation may be a useful therapeutic approach to alleviate a broad scope of diseases in which an uncontrolled inflammatory response plays a major role. METHODS: To enable application of the approach in clinical setting, which requires the genetic modification of the patient's own Tregs, we describe here a novel protocol that allows the efficient retroviral transduction and 2,4,6-trinitrophenol-specific expansion of murine naturally occurring regulatory T cells (nTregs), with a 2,4,6-trinitrophenol-specific tripartite chimeric receptor. RESULTS: Transduced Tregs maintained their Foxp3 level, could undergo repeated expansion upon ex vivo encounter with their cognate antigen in a major histocompatibility complex-independent, costimulation-independent, and contact-dependent manner and specifically suppressed Teff cells. Adoptive transfer of small numbers of the transduced nTregs was associated with antigen-specific, dose-dependent amelioration of trinitrobenzenesulphonic acid colitis. CONCLUSIONS: This study demonstrates that nTregs can be efficiently transduced to express functional, antigen-specific chimeric receptors that enable the specific suppression of effector T cells both in vitro and in vivo. This approach may enable future cell-based therapeutic application in inflammatory bowel disease, as well as other inflammatory disorders.

Treatment of Multiple Myeloma Using Chimeric Antigen Receptor T Cells with Dual Specificity.

Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable successes in fighting B-cell leukemias/lymphomas. Promising response rates are reported in patients treated with B-cell maturation antigen (BCMA) CAR T cells for multiple myeloma. However, responses appear to be nondurable, highlighting the need to expand the repertoire of multiple myeloma-specific targets for immunotherapy and to generate new CAR T cells. Here, we developed a "dual-CAR" targeting two multiple myeloma-associated antigens and explored its safety and efficacy. To reduce the "off-target" toxicity, we used the recognition of paired antigens that were coexpressed by the tumor to induce efficient CAR T-cell activation. The dual-CAR construct presented here was carefully designed to target the multiple myeloma-associated antigens, taking into consideration the distribution of both antigens on normal human tissues. Our results showed that the CD138/CD38-targeted dual CAR (dCAR138-38) elicited a potent anti-multiple myeloma response both in vitro and in vivo NSG mice transplanted with a multiple myeloma cell line and treated with dCAR138-38 showed median survival of 97 days compared with 31 days in the control group treated with mock-lymphocytes. The dCAR138-38 showed increased specificity toward cells expressing both targeted antigens compared with single-antigen-expressing cells and low activity toward primary cells from healthy tissues. Our findings indicated that the dCAR138-38 may provide a potent and safe alternative therapy for patients with multiple myeloma.

Interspecies comparison of prostate cancer gene-expression profiles reveals genes associated with aggressive tumors.

Prostate cancer (PC) is a heterogeneous disease whose aggressive phenotype is the second leading cause of cancer-related death in men. The identification of key molecules and pathways that play a pivotal role in PC progression towards an aggressive form is crucial. A major effort towards this end has been taken by global analyses of gene expression profiles. However, the large body of data did not provide a definitive idea about the genes which are associated with the aggressive growth of PC. In order to identify such genes, we performed an interspecies comparison between several human data sets and high quality microarray data that we generated from the transgenic adenocarcinoma of mouse prostate (TRAMP) strain. The TRAMP PC mimics the histological and pathological appearance as well as the aggressive phenotype of human PC (huPC). Analysis of the microarray data, derived from microdissected TRAMP specimens removed at different stages of the disease yielded genetic signatures delineating the TRAMP PC development and progression. Comparison of the TRAMP data with a set of genes representing the core expression signature of huPC yielded a limited set genes. Some of these genes are known predictors of poor prognosis in huPC. Interestingly, the modulation of genes responsible for the invasive phenotype of huPC occurs in TRAMP already during the transition to prostate intraepithelial neoplasia (PIN) and onwards to localized tumors. We therefore suggest that critical oncogenic events leading to an aggressive phenotype of huPC can be studied in the PIN stage of TRAMP.

Redirection of regulatory T cells with predetermined specificity for the treatment of experimental colitis in mice.

BACKGROUND & AIMS: Treatment with ex vivo expanded regulatory T cells (Tregs) is regarded as a promising therapeutic approach in inflammatory bowel disease but is hampered by impaired Treg accumulation and function at inflammatory sites. We aim to study whether antigen-specific redirected Tregs can overcome these limitations. METHODS: We developed transgenic mice whose T cells, including Tregs, express chimeric receptor (CR) made of antibody variable region as recognition unit and T-cell stimulatory and costimulatory domains to activate specifically in response to the predetermined model antigen 2,4,6-trinitrophenol (TNP). RESULTS: TNP-specific CR-bearing Tregs were potently and specifically activated by exogenous TNP and suppressed effector T cells in the absence of costimulatory B7-CD28 interaction. TNP-specific transgenic (Tg) mice were resistant to 2,4,6-trinitrobenzene sulphonic acid (TNBS) colitis but not to other hapten-mediated colitis. Adoptive transfer of CR-bearing Tregs to wild-type mice with TNBS colitis was associated with significant amelioration of colitis and improved survival. Although TNP-specific CR-bearing Tregs did not suppress oxazolone colitis, they cured it after addition of traces of TNBS to oxazolone-inflamed colons, demonstrating a "bystander" effect. In vivo imaging of adoptively transferred CR-bearing Tregs demonstrated that they preferentially migrate to TNBS-induced colonic mucosal lesions within hours of induction of colitis. CONCLUSIONS: Tregs can be redirected with specificity distinct from that of pathogenic lymphocytes, accumulate at colonic inflammatory lesions, and suppress effector T cells in a specific, nonmajor histocompatibility complex-restricted, and noncostimulatory-dependent manner, resulting in significant amelioration of colitis. Hopefully, this approach will lead to a novel therapy for inflammatory bowel disease, as well as other inflammatory diseases.

Adoptive immunotherapy of prostate cancer bone lesions using redirected effector lymphocytes.

Prostate cancer is currently the most commonly diagnosed noncutaneous malignancy in American men. When metastatic, usually to the bone, the disease is no longer curable and is usually treated palliatively with androgen ablation. However, after conversion to androgen-independent disease, there is no effective therapy currently available. The "T body" approach, which uses genetically reprogrammed lymphocytes derived from the patient and expressing chimeric receptor genes, combines the effector functions of T lymphocytes and NK cells with the ability of antibodies to recognize predefined surface antigens with high specificity and in a non-MHC-restricted manner. We show here the therapeutic efficacy of human lymphocytes bearing erbB2-specific chimeric receptors on human prostate cancer BM lesions in a SCID mouse model after conditioning of the recipient to allow homing and persistent functioning of the adoptively transferred cells. Induction of stromal cell-derived factor-1 production within the BM using low-dose irradiation or cyclophosphamide combined with IL-2 administration enhanced the homing of systemically delivered T bodies, resulting in decreased tumor growth and prostate-specific antigen secretion, prolongation of survival, and even cure of the treated mice. These preclinical studies strongly support the idea that the T body approach has therapeutic potential in disseminated prostate cancer.

Immuno-gene therapy of established prostate tumors using chimeric receptor-redirected human lymphocytes.

Targeted adoptive immunotherapy is an attractive option for prostate cancer given its accessible primary location, the presence of specific tissue and tumor antigens, and the acceptability of collateral destruction of healthy prostrate tissue. The "T-body" approach, which uses genetically programmed, patient-derived lymphocytes transfected with chimeric receptor genes, combines the effector functions of T lymphocytes and natural killer cells with the ability of antibodies to recognize predefined surface antigens with high specificity and in a non-MHC restricted manner. We evaluated the therapeutic efficacy of anti-erbB2 chimeric receptor-bearing human lymphocytes on human prostate cancer xenografts in a SCID mouse model. Local delivery of erbB2-specific T bodies to well-established s.c. and orthotopic tumors, together with systemic administration of interleukin-2, resulted in retardation of both tumor growth and prostate-specific antigen secretion, prolongation of survival, and complete tumor elimination in a significant number of mice. These preclinical studies demonstrate the therapeutic potential of the T-body approach for locally advanced or recurrent prostate cancer as an adjunct to, or after, conventional therapy.

The emergence of T-bodies/CAR T cells.

The nickname "T-body" is used to denote a T cell expressing an antigen-specific or antibody-based chimeric receptor that combines antibody specificity with T-cell effector or regulatory function. Initially, we designed and constructed chimeric antibody-based receptors and expressed them in T cells to study the role of major histocompatibility complex in triggering T-cell activation. To this end, we replaced both variable domains (Vα and Vß of the native T-cell receptor chains) with antibody-derived VH and VL sequences. After transfection into T cells, the 2 chimeric chains paired, associated with the CD3 complex, and endowed transfectants with non-major histocompatibility complex-restricted antibody type specificity. In subsequent studies, we developed next generation of chimeric antibody-based receptors by fusing an antibody single-chain variable fragment to T-cell activation motifs. This modular configuration simplified gene transfer, avoided mixed pairing with endogenous T-cell receptor chains, and enabled simultaneous insertion of various domains as costimulatory moieties to generate T-bodies with efficient antitumor reactivity.

Irradiation enhances the metastatic potential of prostatic small cell carcinoma xenografts.

BACKGROUND: Small cell carcinoma of the prostate (SCCP) is a rare subset of prostate cancer (0.5-2% of all prostatic carcinomas), predominantly composed of neuroendocrine (NE) cells, with a very poor prognosis. Irradiation is one of the mainstay options for SCCP local treatment, yet, little is known about the clinical response of these aggressive tumors to radiotherapy. METHODS: Using SCID mice, the response to fractionated ionizing radiation (IR) of two unique human NE xenografts of SCCP (WISH-PC2 and WM-4A) was investigated. RESULTS: Fractionated irradiation of WISH-PC2 xenografts using total doses of >24 Gy induced a delay in tumor growth, while total doses of >36 Gy led to local tumor eradication. However, most of the irradiated mice suffered from disseminated metastases. Similarly, in the WM-4A xenograft, a total dose of 20 Gy led to tumor growth delay and some of the mice also developed metastases. Non-irradiated local xenografts failed to disseminate, even following surgical excision of the main tumor mass; however, tumor cells administered intravenously did form metastases. Metastases of both xenografts were located in the adrenal/kidney and inter-scapular regions, areas rich in brown adipose tissue. A correlation was found between the appearance of irradiation-induced metastases and activation of the gelatinase activity of matrix metalloproteinase-9. CONCLUSIONS: Clinically, this study raises the possibility that radiation to SCCP may promote metastatic disease. For patients in whom prostate biopsy shows a predominance of small cell cancer, it may be necessary to deliver systemic therapy together with the radiotherapy in order to prevent the development of metastases.