Mobilization of innate and adaptive antitumor immune responses by the RNP-targeting antibody ATRC-101.

Immunotherapy approaches focusing on T cells have provided breakthroughs in treating solid tumors. However, there remains an opportunity to drive anticancer immune responses via other cell types, particularly myeloid cells. ATRC-101 was identified via a target-agnostic process evaluating antibodies produced by the plasmablast population of B cells in a patient with non-small cell lung cancer experiencing an antitumor immune response during treatment with checkpoint inhibitor therapy. Here, we describe the target, antitumor activity in preclinical models, and data supporting a mechanism of action of ATRC-101. Immunohistochemistry studies demonstrated tumor-selective binding of ATRC-101 to multiple nonautologous tumor tissues. In biochemical analyses, ATRC-101 appears to target an extracellular, tumor-specific ribonucleoprotein (RNP) complex. In syngeneic murine models, ATRC-101 demonstrated robust antitumor activity and evidence of immune memory following rechallenge of cured mice with fresh tumor cells. ATRC-101 increased the relative abundance of conventional dendritic cell (cDC) type 1 cells in the blood within 24 h of dosing, increased CD8+ T cells and natural killer cells in blood and tumor over time, decreased cDC type 2 cells in the blood, and decreased monocytic myeloid-derived suppressor cells in the tumor. Cellular stress, including that induced by chemotherapy, increased the amount of ATRC-101 target in tumor cells, and ATRC-101 combined with doxorubicin enhanced efficacy compared with either agent alone. Taken together, these data demonstrate that ATRC-101 drives tumor destruction in preclinical models by targeting a tumor-specific RNP complex leading to activation of innate and adaptive immune responses.

Non-progressing cancer patients have persistent B cell responses expressing shared antibody paratopes that target public tumor antigens.

There is significant debate regarding whether B cells and their antibodies contribute to effective anti-cancer immune responses. Here we show that patients with metastatic but non-progressing melanoma, lung adenocarcinoma, or renal cell carcinoma exhibited increased levels of blood plasmablasts. We used a cell-barcoding technology to sequence their plasmablast antibody repertoires, revealing clonal families of affinity matured B cells that exhibit progressive class switching and persistence over time. Anti-CTLA4 and other treatments were associated with further increases in somatic hypermutation and clonal family size. Recombinant antibodies from clonal families bound non-autologous tumor tissue and cell lines, and families possessing immunoglobulin paratope sequence motifs shared across patients exhibited increased rates of binding. We identified antibodies that caused regression of, and durable immunity toward, heterologous syngeneic tumors in mice. Our findings demonstrate convergent functional anti-tumor antibody responses targeting public tumor antigens, and provide an approach to identify antibodies with diagnostic or therapeutic utility.

NKG2D-deficient mice are defective in tumor surveillance in models of spontaneous malignancy.

Ligands for the NKG2D stimulatory receptor are frequently upregulated on tumor lines, rendering them sensitive to natural killer (NK) cells, but the role of NKG2D in tumor surveillance has not been addressed in spontaneous cancer models. Here, we provided the first characterization of NKG2D-deficient mice, including evidence that NKG2D was not necessary for NK cell development but was critical for immunosurveillance of epithelial and lymphoid malignancies in two transgenic models of de novo tumorigenesis. In both models, we detected NKG2D ligands on the tumor cell surface ex vivo, providing needed evidence for ligand expression by primary tumors. In a prostate cancer model, aggressive tumors arising in NKG2D-deficient mice expressed higher amounts of NKG2D ligands than did similar tumors in wild-type mice, suggesting an NKG2D-dependent immunoediting of tumors in this model. These findings provide important genetic evidence for surveillance of primary tumors by an NK receptor.

Cell-intrinsic abrogation of TGF-ß signaling delays but does not prevent dysfunction of self/tumor-specific CD8 T cells in a murine model of autochthonous prostate cancer.

Adoptive T cell therapy (ACT) for the treatment of established cancers is actively being pursued in clinical trials. However, poor in vivo persistence and maintenance of antitumor activity of transferred T cells remain major problems. TGF-ß is a potent immunosuppressive cytokine that is often expressed at high levels within the tumor microenvironment, potentially limiting T cell-mediated antitumor activity. In this study, we used a model of autochthonous murine prostate cancer to evaluate the effect of cell-intrinsic abrogation of TGF-ß signaling in self/tumor-specific CD8 T cells used in ACT to target the tumor in situ. We found that persistence and antitumor activity of adoptively transferred effector T cells deficient in TGF-ß signaling were significantly improved in the cancerous prostate. However, over time, despite persistence in peripheral lymphoid organs, the numbers of transferred cells in the prostate decreased and the residual prostate-infiltrating T cells were no longer functional. These findings reveal that TGF-ß negatively regulates the accumulation and effector function of transferred self/tumor-specific CD8 T cells and highlight that, when targeting a tumor Ag that is also expressed as a self-protein, additional substantive obstacles are operative within the tumor microenvironment, potentially hampering the success of ACT for solid tumors.

A gene signature identified using a mouse model of androgen receptor-dependent prostate cancer predicts biochemical relapse in human disease.

Mutations in the androgen receptor (AR) have been detected in experimental and clinical prostate tumors. Mice with enforced prostate-specific expression of one such receptor variant, AR-E231G, invariably develop prostatic intraepithelial neoplasia by 12 weeks and metastatic prostate cancer by 52 weeks. The aim of this study was to identify genes with altered expression in the prostates of AR-E231G mice at an early stage of disease that may act as drivers of AR-mediated tumorigenesis. The gene expression profile of AR-E231G prostate tissue from 12-week-old mice was compared to an equivalent profile from mice expressing the AR-T857A receptor variant (analogous to the AR-T877A variant in LNCaP cells), which do not develop prostate tumors. One hundred and thirty-two genes were differentially expressed in AR-E231G prostates. Classification of these genes revealed enrichment for cellular pathways known to be involved in prostate cancer, including cell cycle and lipid metabolism. Suppression of two genes upregulated in the AR-E231G model, ADM and CITED1, increased cell death and reduced proliferation of human prostate cancer cells. Many genes differentially expressed in AR-E231G prostates are also deregulated in human tumors. Three of these genes, ID4, NR2F1 and PTGDS, which were expressed at consistently lower levels in clinical prostate cancer compared to nonmalignant tissues, formed a signature that predicted biochemical relapse (hazard ratio 2.2, p = 0.038). We believe that our findings support the value of this novel mouse model of prostate cancer to identify candidate therapeutic targets and/or biomarkers of human disease.

Cutting Edge: Tumor-specific CD8+ T cells infiltrating prostatic tumors are induced to become suppressor cells.

We previously reported that naive, tumor-specific CD8(+) (TcR-I) T cells transferred into prostate tumor-bearing mice traffic to the prostate where they become tolerized. We now report that TcR-I cells suppress the proliferation of naive T cells. This suppression is mediated at least in part by secreted factors, and the suppressive activity can be blocked by Abs directed against TGF-beta. We further report that TcR-I cells must infiltrate the prostate to acquire suppressive activity. Delivery of tumor-specific CD4(+) T cells prevents the conversion of TcR-I cells into suppressor cells. Taken together, our findings may have critical implications for sustaining T cell responsiveness during immunotherapy, as the development of suppressor cells in the tumor microenvironment may eliminate the potency of T cells primed in the periphery or delivered during adoptive immunotherapy.

SPAS-1 (stimulator of prostatic adenocarcinoma-specific T cells)/SH3GLB2: A prostate tumor antigen identified by CTLA-4 blockade.

Discovery of immunologically relevant antigens in prostate cancer forms the basis for developing more potent active immunotherapy. We report here a strategy using the transgenic adenocarcinoma of mouse prostate (TRAMP) model, which allows for the functional identification of immunogenic prostate tumor antigens with relevance for human immunotherapy. Using a combination of active tumor vaccination in the presence of CTL-associated antigen 4 (CTLA-4) in vivo blockade, we elicited tumor-specific T cells used to expression clone the first T cell-defined TRAMP tumor antigen, called Spas-1 (stimulator of prostatic adenocarcinoma specific T cells-1). Spas-1 expression was increased in advanced primary TRAMP tumors. We show that the immunodominant SPAS-1 epitope SNC9-H(8) arose from a point mutation in one allele of the gene in TRAMP tumor cells, and that immunization with dendritic cells pulsed with SNC9-H(8) peptide resulted in protection against TRAMP-C2 tumor challenge. In humans, the Spas-1 ortholog SH3GLB2 has been reported to be overexpressed in prostate cancer metastases. Additionally, we identified a nonmutated HLA-A2-binding epitope in the human ortholog SH3GLB2, which primed T cells from healthy HLA-A2(+) individuals in vitro. Importantly, in vitro-primed T cells also recognized naturally processed and presented SH3GLB2. Our findings demonstrate that our in vivo CTLA-4 blockade-based T cell expression cloning can identify immunogenic cancer antigens with potential relevance for human immunotherapy.

Genetic ablation of the amplified-in-breast cancer 1 inhibits spontaneous prostate cancer progression in mice.

Although the amplified-in-breast cancer 1 (AIB1; SRC-3, ACTR, or NCoA3) was defined as a coactivator for androgen receptor (AR) by in vitro studies, its role in AR-mediated prostate development and prostate cancer remained unexplored. We report here that AIB1 is expressed in the basal and stromal cells but not in the epithelial cells of the normal mouse prostates. AIB1 deficiency only slightly delayed prostate growth and had no effect on androgen-dependent prostate regeneration, suggesting an unessential role of AIB1 in AR function in the prostate. Surprisingly, when prostate tumorigenesis was induced by the SV40 transgene in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, AIB1 expression was observed in certain epithelial cells of the prostate intraepithelial neoplasia (PIN) and well-differentiated carcinoma and in almost all cells of the poorly differentiated carcinoma. After AIB1 was genetically inactivated in AIB1-/-/TRAMP mice, the progression of prostate tumorigenesis in most AIB1-/-/TRAMP mice was arrested at the well-differentiated carcinoma stage. Wild-type (WT)/TRAMP mice developed progressive, multifocal, and metastatic prostate tumors and died between 25 and 34 weeks. In contrast, AIB1-/-/TRAMP mice only exhibited PIN and early-stage well-differentiated carcinoma by 39 weeks. AIB1-/-/TRAMP prostates showed much lower cell proliferation than WT/TRAMP prostates. Most AIB1-/-/TRAMP mice could survive more than 35 weeks and died with other types of tumors or unknown reasons. Our results indicate that induction of AIB1 expression in partially transformed epithelial cells is essential for progression of prostate tumorigenesis into poorly differentiated carcinoma. Inhibition of AIB1 expression or function in the prostate epithelium may be a potential strategy to suppress prostate cancer initiation and progression.

Functional neoangiogenesis imaging of genetically engineered mouse prostate cancer using three-dimensional power Doppler ultrasound.

We report the first application of high-frequency three-dimensional power Doppler ultrasound imaging in a genetically engineered mouse (GEM) prostate cancer model. We show that the technology sensitively and specifically depicts functional neoangiogenic blood flow because little or no flow is measurable in normal prostate tissue or tumors smaller than 2-3 mm diameter, the neoangiogenesis "switch-on" size. Vascular structures depicted by power Doppler were verified using Microfil-enhanced micro-computed tomography (micro-CT) and by correlation with microvessel distributions measured by immunohistochemistry and enhanced vascularity visualized by confocal microscopy in two GEM models [transgenic adenocarcinoma of the mouse prostate (TRAMP) and PSP94 gene-directed transgenic mouse adenocarcinoma of the prostate (PSP-TGMAP)]. Four distinct phases of neoangiogenesis in cancer development were observed, specifically, (a) an early latent phase; (b) establishment of a peripheral capsular vascular structure as a neoangiogenesis initiation site; (c) a peak in tumor vascularity that occurs before aggressive tumor growth; and (d) rapid tumor growth accompanied by decreasing vascularity. Microsurgical interventions mimicking local delivery of antiangiogenesis drugs were done by ligating arteries upstream from feeder vessels branching to the prostate. Microsurgery produced an immediate reduction of tumor blood flow, and flow remained low from 1 h to 2 weeks or longer after treatment. Power Doppler, in conjunction with micro-CT, showed that the tumors recruit secondary blood supplies from nearby vessels, which likely accounts for the continued growth of the tumors after surgery. The microsurgical model represents an advanced angiogenic prostate cancer stage in GEM mice corresponding to clinically defined hormone-refractory prostate cancer. Three-dimensional power Doppler imaging is completely noninvasive and will facilitate basic and preclinical research on neoangiogenesis in live animal models.

Tumor paint: a chlorotoxin:Cy5.5 bioconjugate for intraoperative visualization of cancer foci.

Toward the goal of developing an optical imaging contrast agent that will enable surgeons to intraoperatively distinguish cancer foci from adjacent normal tissue, we developed a chlorotoxin:Cy5.5 (CTX:Cy5.5) bioconjugate that emits near-IR fluorescent signal. The probe delineates malignant glioma, medulloblastoma, prostate cancer, intestinal cancer, and sarcoma from adjacent non-neoplastic tissue in mouse models. Metastatic cancer foci as small as a few hundred cells were detected in lymph channels. Specific binding to cancer cells is facilitated by matrix metalloproteinase-2 (MMP-2) as evidenced by reduction of CTX:Cy5.5 binding in vitro and in vivo by a pharmacologic blocker of MMP-2 and induction of CTX:Cy5.5 binding in MCF-7 cells following transfection with a plasmid encoding MMP-2. Mouse studies revealed that CTX:Cy5.5 has favorable biodistribution and toxicity profiles. These studies show that CTX:Cy5.5 has the potential to fundamentally improve intraoperative detection and resection of malignancies.

EZC-prostate models offer high sensitivity and specificity for noninvasive imaging of prostate cancer progression and androgen receptor action.

In vivo imaging advances have greatly expanded the use of animal cancer models. Herein, we describe two new models that permit prostate imaging ex vivo, in vivo, and in utero. Further, we show the use of these models for detecting small metastasis and testing reagents that modulate the androgen receptor (AR) axis. A luciferase reporter gene was directed to the prostate epithelium using three composite promoters called human kallikrein 2 (hK2)-E3/P, PSA-E2/P, and ARR2PB, derived from hK2, PSA, and rat probasin regulatory elements, to generate the EZC1, EZC2, and EZC3-prostate mice, respectively. EZC2 and EZC3-prostate display robust expression in the prostate with only minimal detectable expression in other organs, including testes and epididymis. Luciferase expression was detected as early as embryonic day 13 (E13) in the urogenital track. To image prostate cancer progression, lines of EZC mice were bred with prostate cancer models TRAMP and JOCK1, and imaged longitudinally. When crossed with prostate cancer models, EZC3 facilitated detection of metastatic lesions although total prostate luciferase expression was static or reduced due to weakening of AR-regulated promoters. Castration reduced luciferase expression by 90% and 97% in EZC2 and EZC3 mice, respectively, and use of GnRH antagonist also led to extensive inhibition of reporter activity. The EZC-prostate model permits prostate imaging in vivo and should be useful for imaging prostate development, growth, metastasis, and response to treatment noninvasively and longitudinally. These models also provide powerful new reagents for developing improved drugs that inhibit the AR axis.

Targeting Aurora kinases for the treatment of prostate cancer.

Inappropriate expression of the Aurora kinases can induce aberrant mitosis, centrosome irregularities, and chromosomal instability, which lead to anueploidy and cell transformation. Here, we report that Aurora-A and Aurora-B are highly expressed in primary human and mouse prostate cancers and prostate cancer cell lines. In clinical samples, levels of Aurora-A and Aurora-B were significantly elevated in prostatic intraepithelial neoplasia lesions and prostate tumors when compared with the non-neoplastic samples. Interestingly, expression of Aurora-A in non-neoplastic prostates correlated with seminal vesicle invasion (rho = 0.275, P = 0.0169) and in prostate tumor with positive surgical margins (rho = 0.265, P = 0.0161). In addition, nuclear expression of Aurora-B in prostatic intraepithelial neoplasia lesions correlated with clinical staging of the tumor (rho = -0.4, P = 0.0474) whereas cytoplasmic expression in tumors correlated with seminal vesicle invasion (rho = 0.282, P = 0.0098). Cell lines and primary tumors derived from the TRAMP model were also found to express high levels of Aurora-A and Aurora-B. When human PC3, LNCaP, and mouse C1A cells were treated with the potent Aurora kinase inhibitor VX680, which attenuates phosphorylation of histone H3, cancer cell survival was reduced. VX680 could further reduce cell viability >2-fold when used in combination with the chemotherapy drug doxorubicin. Our findings support a functional relationship between Aurora kinase expression and prostate cancer and the application of small-molecule inhibitors in therapeutic modalities.

Identification of genes potentially involved in the acquisition of androgen-independent and metastatic tumor growth in an autochthonous genetically engineered mouse prostate cancer model.

BACKGROUND: A major focus of prostate cancer research has been to identify genes that are deregulated during tumor progression, potentially providing diagnostic markers and therapeutic targets. METHODS: We have employed serial analysis of gene expression (SAGE) and microarray hybridization to identify alterations that occur during malignant transformation in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model. Many of these alterations were validated by real-time PCR (rtPCR). RESULTS: We identified several hundred mRNAs that were deregulated. Cluster analysis of microarray profiles with samples from various stages of the disease demonstrated that androgen-independent (AI) primary tumors are similar to metastases; 180 transcripts have expression patterns suggesting an involvement in the genesis of late-stage tumors, and our data support a role for phospholipase A2 group IIA in the acquisition of their highly aggressive characteristics. CONCLUSIONS: Our analyses identified well-characterized genes that were previously known to be involved in prostate cancer, validating our study, and also uncovered transcripts that had not previously been implicated in prostate cancer progression.

Bub1 up-regulation and hyperphosphorylation promote malignant transformation in SV40 tag-induced transgenic mouse models.

Rodents do not naturally develop prostate cancer. Currently, most widely used genetically engineered mouse prostate cancer models use SV40 T/tag oncogene. To understand the mechanism underlying prostate cancer development in transgenic and knock-in SV40 Tag mouse models, we did cDNA microarray analyses, comparing gene expression profiles of prostate cancer tissues from early-, late-, and advance-stage androgen-independent prostate cancers. Of the 67 genes that were up-regulated by > or = 10-fold, 40 are known to be required for chromosome stability. In particular, the spindle checkpoint component Bub1 was persistently up-regulated from early to advanced androgen-independent prostate cancer lesions. Significantly, Bub1, which is required for accurate chromosome segregation during mitosis, has recently been reported to bind SV40 Tag. Consistent with a spindle checkpoint defect, flow cytometry experiments indicate that advanced androgen-independent prostate cancer tumors exhibit aneuploidy, along with up-regulation of levels of both Bub1 mRNA and Bub1 protein or hyperphosphorylation. Importantly, up-regulation and hyperphosphorylation of Bub1 were also observed in established human prostate cancer cell lines and in clinical studies. Furthermore, analysis of human prostate cancer lines showed impaired spindle checkpoint function and endoreduplication following exposure to spindle toxins. Small interfering RNA-mediated repression of Bub1 in the human prostate cancer line PC-3 restrained cell proliferation, an effect mimicked by inhibition of mitogen-activated protein kinase, an upstream activator of Bub1. Thus, by perturbing Bub1 function, our observations suggest a new mechanism whereby the SV40 Tag oncoprotein promotes chromosomal instability and aneuploidy in transgenic mouse prostate cancer models. Whereas the exact details of this mechanism remain unclear, our novel findings raise the possibility of exploiting Bub1 as a new therapeutic target in the treatment of prostate cancer, the most common cancer in adult men in North America.

Pten dose dictates cancer progression in the prostate.

Complete inactivation of the PTEN tumor suppressor gene is extremely common in advanced cancer, including prostate cancer (CaP). However, one PTEN allele is already lost in the vast majority of CaPs at presentation. To determine the consequence of PTEN dose variations on cancer progression, we have generated by homologous recombination a hypomorphic Pten mouse mutant series with decreasing Pten activity: Pten(hy/+) > Pten(+/-) > Pten(hy/-) (mutants in which we have rescued the embryonic lethality due to complete Pten inactivation) > Pten prostate conditional knockout (Pten(pc)) mutants. In addition, we have generated and comparatively analyzed two distinct Pten(pc) mutants in which Pten is inactivated focally or throughout the entire prostatic epithelium. We find that the extent of Pten inactivation dictate in an exquisite dose-dependent fashion CaP progression, its incidence, latency, and biology. The dose of Pten affects key downstream targets such as Akt, p27(Kip1), mTOR, and FOXO3. Our results provide conclusive genetic support for the notion that PTEN is haploinsufficient in tumor suppression and that its dose is a key determinant in cancer progression.

Breast cancer resistance protein-mediated efflux of androgen in putative benign and malignant prostate stem cells.

Malignantly transformed stem cells represent a potential common nidus for the primary cancer and the recurrent cancer that arises after treatment failure. Putative prostate stem cells and prostate tumor stem cells in benign and malignant human prostate tissue, in primary human prostate xenografts, and in the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model of prostate cancer, are defined by expression of breast cancer resistance protein (BCRP), a marker of pluripotent hematopoietic, muscle, and neural stem cells, and by an absence of androgen receptor (AR) protein. Inhibition of BCRP-mediated efflux of dihydrotestosterone by novobiocin or fumitremorgin C in a rat prostate progenitor cell line that expresses BCRP and AR mRNAs, but minimal AR protein, results in stabilization and nuclear translocation of AR protein, providing a mechanism for lack of AR protein in BCRP-expressing stem cells. In both benign and malignant human prostate tissue, the rare epithelial cells that express BCRP and lack AR protein are localized in the basal cell compartment, survive androgen deprivation, and maintain proliferative potential in the hypoxic, androgen-deprived prostate. Putative prostate tumor stem cells that express BCRP but not AR protein in TRAMP are the source of a BCRP-negative and AR-negative, Foxa2- and SV40Tag-expressing, transit amplifying compartment that progresses to the poorly differentiated carcinomas that arise rapidly after castration. Therefore, BCRP expression isolates prostate stem/tumor stem cells from the prostate tissue microenvironment through constitutive efflux of androgen, protecting the putative tumor stem cells from androgen deprivation, hypoxia, or adjuvant chemotherapy, and providing the nidus for recurrent prostate cancer.

Adoptive transfer of tumor-reactive transforming growth factor-beta-insensitive CD8+ T cells: eradication of autologous mouse prostate cancer.

Transforming growth factor (TGF)-beta is a potent immunosuppressant. Overproduction of TGF-beta by tumor cells may lead to tumor evasion from the host immune surveillance and tumor progression. The present study was conducted to develop a treatment strategy through adoptive transfer of tumor-reactive TGF-beta-insensitive CD8+ T cells. The mouse TRAMP-C2 prostate cancer cells produced large amounts of TGF-beta1 and were used as an experimental model. C57BL/6 mice were primed with irradiated TRAMP-C2 cells. CD8+ T cells were isolated from the spleen of primed animals, were expanded ex vivo, and were rendered TGF-beta insensitive by infecting with a retrovirus containing dominant-negative TGF-beta type II receptor. Results of in vitro cytotoxic assay revealed that these CD8+ T cells showed a specific and robust tumor-killing activity against TRAMP-C2 cells but were ineffective against an irrelevant tumor line, B16-F10. To determine the in vivo antitumor activity, recipient mice were challenged with a single injection of TRAMP-C2 cells for a period up to 21 days before adoptive transfer of CD8+ T cells was done. Pulmonary metastasis was either eliminated or significantly reduced in the group receiving adoptive transfer of tumor-reactive TGF-beta-insensitive CD8+ T cells. Results of immunofluorescent studies showed that only tumor-reactive TGF-beta-insensitive CD8+ T cells were able to infiltrate into the tumor and mediate apoptosis in tumor cells. Furthermore, transferred tumor-reactive TGF-beta-insensitive CD8+ T cells were able to persist in tumor-bearing hosts but declined in tumor-free animals. These results suggest that adoptive transfer of tumor-reactive TGF-beta-insensitive CD8+ T cells may warrant consideration for cancer therapy.

beta1A integrin expression is required for type 1 insulin-like growth factor receptor mitogenic and transforming activities and localization to focal contacts.

The cells' ability to proliferate in response to growth factor stimulation is significantly altered during cancer progression. To investigate the mechanisms underlying these alterations in prostate cancer, the role and expression of beta1A integrin and type 1 insulin-like growth factor receptor (IGF-IR), known to contribute to cell proliferation and transformation, were analyzed. Using small interfering RNA oligonucleotides to down-regulate beta1A, we show that beta1A expression is required for IGF-IR-mediated prostate cancer cell proliferation and anchorage-independent growth. In vivo, using age-matched transgenic adenocarcinoma of mouse prostate (TRAMP) mice at different stages of prostate cancer [prostatic intraepithelial neoplasia, PIN; well-differentiated adenocarcinoma, WD; and poorly differentiated adenocarcinoma, PD], the expression of beta1A and of IGF-IR was studied. beta1A and IGF-IR expression levels were concurrently up-regulated in high PIN and WD, whereas their expression did not correlate in late-stage PD. In contrast to the up-regulated expression of beta1A, the levels of beta1C, a beta1 cytoplasmic variant that inhibits cell proliferation, were down-regulated in all stages of prostate cancer. A similar expression pattern was observed for a beta1C downstream effector, Grb2-associated binder-1 (Gab1) which is known to inhibit IGF-IR phosphorylation. To analyze in vitro the mechanistic implications of beta1A, beta1C, and Gab1 deregulation in prostate cancer, we investigated whether expression of either beta1 variant in beta1-null cells affected IGF-IR localization. We found that IGF-IR and beta1A were colocalized in highly specialized integrin signaling compartments, designated focal contacts. However, in the presence of beta1C, IGF-IR remained diffuse on the cell surface and did not localize to focal contacts. The findings that beta1 integrins and IGF-IR are concurrently deregulated and that expression of beta1 integrins is necessary to achieve appropriate IGF-IR intracellular distribution point to the important role that the cross-talk between these receptors may have during prostate cancer progression and will be helpful in formulating new therapeutic strategies.

A new three-dimensional ultrasound microimaging technology for preclinical studies using a transgenic prostate cancer mouse model.

Prostate cancer is the most common cancer in adult men in North America. Preclinical studies of prostate cancer employ genetically engineered mouse models, because prostate cancer does not occur naturally in rodents. Widespread application of these models has been limited because autopsy was the only reliable method to evaluate treatment efficacy in longitudinal studies. This article reports the first use of three-dimensional ultrasound microimaging for measuring tumor progression in a genetically engineered mouse model, the 94-amino acid prostate secretory protein gene-directed transgenic prostate cancer model. Qualitative comparisons of three-dimensional ultrasound images with serial histology sections of prostate tumors show the ability of ultrasound to accurately depict the size and shape of malignant masses in live mice. Ultrasound imaging identified tumors ranging from 2.4 to 14 mm maximum diameter. The correlation coefficient of tumor diameter measurements done in vivo with three-dimensional ultrasound and at autopsy was 0.998. Prospective tumor detection sensitivity and specificity were both >90% when diagnoses were based on repeated ultrasound examinations done on separate days. Representative exponential growth curves constructed via longitudinal ultrasound imaging indicated volume doubling times of 5 and 13 days for two prostate tumors. Compared with other microimaging and molecular imaging modalities, the application of three-dimensional ultrasound imaging to prostate cancer in mice showed advantages, such as high spatial resolution and contrast in soft tissue, fast and uncomplicated protocols, and portable and economical equipment that will likely enable ultrasound to become a new microimaging modality for mouse preclinical trial studies.

Blockade of transforming growth factor-{beta} signaling in tumor-reactive CD8(+) T cells activates the antitumor immune response cycle.

Transforming growth factor-beta (TGF-beta) is a potent immunosuppressant. Overproduction of TGF-beta by tumor cells leads to evasion of host immune surveillance and tumor progression. Results of our early studies showed that adoptive transfer of tumor-reactive, TGF-beta-insensitive CD8(+) T cells into immunocompetent mice was able to eradicate lung metastasis of mouse prostate cancer. The present study was conducted with three objectives. (a) We tested if this technology could be applied to the treatment of solid xenograft tumors in allogeneic immunodeficient hosts. (b) We determined relevant variables in the tumor microenvironment with the treatment. (c) We tested if immune cells other than CD8(+) T cells were required for the antitumor effect. Mouse prostate cancer cells, TRAMP-C2 of the C57BL/6 strain, grown in immunodeficient allogeneic hosts of BALB/c strain, were used as a xenograft model. Tumor-reactive CD8(+) T cells from C57BL/6 mice were isolated, expanded ex vivo, and rendered insensitive to TGF-beta by introducing a dominant-negative TGF-beta type II receptor vector. Seven days following s.c. injection of TRAMP-C2 cells (5 x 10(5)) into the flank of male BALB/c-Rag1(-/-) mice, tumor-reactive, TGF-beta-insensitive CD8(+) T cells (1.5 x 10(7)) were transferred with and without the cotransfer of an equal number of CD8-depleted splenocytes from C57BL/6 donors. Naive CD8(+) T cells or green fluorescent protein-empty vector-transfected tumor-reactive CD8(+) T cells were transferred as controls. Forty days following the transfer, the average tumor weight in animals that received cotransfer of tumor-reactive, TGF-beta-insensitive CD8(+) T cells and CD8-depleted splenocytes was at least 50% less than that in animals of all other groups (P < 0.05). Tumors in animals of the former group showed a massive infiltration of CD8(+) T cells. This was associated with secretion of relevant cytokines, decreased tumor proliferation, reduced angiogenesis, and increased tumor apoptosis. Based on these results, we postulated a concept of antitumor immune response cycle in tumor immunology.