Reduced Tumor Area Research Articles

CAR T cells against TRP1 significantly reduce melanoma growth in vitro and in vivo

Abstract CAR T cells have shown great efficacy for patients with liquid malignancies. However, CAR T cell treatments have had limited efficacy in patients with solid lesions, such as melanoma. One reason is the lack of suitable targets specific to the tumor antigen, which also do not cause toxicity. TRP1 is abundantly expressed in melanoma cells as well as in healthy melanocytes. While targeting healthy melanocytes may cause vitiligo, that condition is not life threatening. Previous work shows monoclonal antibodies against TRP1 reject melanoma tumors and lung metastases. Therefore, we designed a CAR with an scFv against human TRP1 along with 41BB and CD3z costimulation. When we examined CAR T cell cytotoxicity in vitro, we found a significant reduction in melanoma target cells after 48hrs co-culture with CAR T cells at 10:1 and 5:1 E:T ratios compared to untransduced (UT) cells. CAR T cell cytokine production after melanoma co-culture also showed significantly higher IFNg compared to UT cells. To examine CAR T cell efficacy in vivo, we challenged NSG mice intradermally with melanoma tumor cells. Four days after the tumor challenge, mice were given CAR T or UT cells by tail vein injection. We found that mice given anti-TRP1 CAR T cells had significantly reduced tumor area by day 10 and had significantly increased survival compared to mice given UT cells. This work shows that TRP1 can be effectively targeted by CAR T cells and increase the survival of mice with melanoma.

Abstract 6406: Radiation and STING activation limit tumor development and modulate the immune environment via distinct mechanisms

Abstract Carcinogen-induced lung cancer in A/J mice is an established model of tumor initiation and development. The impact of radiation, immune response, and STING pathway activation on tumor development in this model is not well understood. In this study, we examined the effect of radiation and the STING agonist, DMXAA (Vadimezan), on the development of urethane-induced lung cancer. A/J mice were treated with urethane (i.p.) to induce the development of lung tumors. Three days after urethane treatment, mice received 13 Gy thorax radiation treatment (RT)/mock RT, DMXAA (20 mg/kg i.p.)/vehicle, or a combination treatment (n = 5). Mice were euthanized after 6 hours to 7 days after treatment to assess early events in the lung with RNA-sequencing and flow cytometry, or 5 months after treatment to assess tumor growth and long-term changes to the immune microenvironment with multiplex immunofluorescence. Mice treated with urethane followed by RT had fewer lung surface tumors (p < 0.01) and reduced tumor area (p = 0.0732) compared to their mock RT controls. In parallel, mice that received urethane pre-treatment and RT had high numbers of tertiary lymphoid structures (TLSs; these contained T-cells, B-cells, MHCII+ APCs and CXCL13) compared to the mock RT control mice (p < 0.001). Analysis of immune cell contents of the TLS revealed a notable increase in CD4, CD8 and regulatory T-cells in the RT condition (with urethane pretreatment). Important early events in the lung following RT of urethane pre-treated mice included a transient increase in type I and type II interferons, CD4 and CD8 T-cell infiltration (p < 0.001) and a strong downregulation of cell cycle events. In contrast to these findings, DMXAA treatment after urethane resulted in fewer tumors (p < 0.05), but larger tumors compared to the vehicle-treated group. An increase in immune cells was observed in the lung, however, without clear structures that resembled TLSs. Early events following DMXAA treatment with urethane pre-treatment included dampened levels of type I IFN and TNF-α in circulation and a decrease in T-cell infiltration into the lungs. Interestingly, RT and DMXAA combination treatment (with urethane pre-treatment) drove a synergistic anti-tumor effect, further reducing tumor numbers compared to the RT or DMXAA controls 5 months after treatment. RT and STING agonist treatment of pre-neoplastic lesions have differential impacts on tumor growth in the A/J mouse model, and work synergistically when combined. The different outcomes of RT and DMXAA on tumor growth may be driven by their distinctive mechanism of action on immune responses and capacities to form TLSs. These findings may have future implications for strategies for the early treatment of lung and other cancers, and they suggest that immune responses, including modulation of the STING pathway, may be an important aspect of early tumor development that could be targeted therapeutically. Citation Format: Kay Shigemori, Yanyan Jiang, Bruno Beernaert, Anderson J. Ryan, Eileen E. Parkes. Radiation and STING activation limit tumor development and modulate the immune environment via distinct mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6406.

Development of an Aryloxazole Derivative as a Brain-Permeable Anti-Glioblastoma Agent.

Glioblastoma drug development has been difficult due to the extremely low blood brain barrier (BBB) penetration of conventional anti-cancer agents. P-glycoprotein, an efflux membrane transporter, is responsible for the poor brain uptake of small and hydrophobic drug substances. To develop brain-penetrable anti-tumor agents, we designed colchicine derivatives containing an aryloxazole moiety, which is known to inhibit P-glycoprotein. Among those tested, an aryloxazole derivative named KIST-G1 showed the strongest anti-glioblastoma cell proliferation activity (IC50 = 3.2 ± 0.8 nM). Compared to colchicine, KIST-G1 showed dramatically increased BBB-permeable properties presenting 51.7 ± 0.5 (10−6 cm/s) parallel artificial membrane permeability assay (PAMPA) permeability and 45.0 ± 6.0% of P-gp inhibition. Aid by the BBB-permeable properties, KIST-G1 (5 mg/kg) suppressed glioblastoma cell growth and migration almost completely in the brain of glioblastoma xenograft models by showing 98.2 ± 0.1% reduced tumor area compared with phosphate buffered saline (PBS)-injected control. In comparison, temozolomide, which is the most widely used drug for glioblastoma, showed only moderate effects. Our results demonstrate the effectiveness of an aryloxazole moiety in targeting brain tumors and suggest KIST-G1 as a potent anti-glioblastoma agent.

Abstract B202: Defining the role of IL-27 signaling on responses to metastatic melanoma

Abstract Checkpoint blockade and cellular therapies for cancer treatment have yielded impressive clinical results. These approaches harness the ability of T-cells, critical mediators of the body’s immune response to infection, to eliminate tumors. Entry of T-cells into the tumor microenvironment exposes them to an array of regulatory signals that impact T-cell differentiation and function. The immunomodulatory cytokine, interleukin 27 (IL-27), produced by tumor cells and immune cells, is a critical regulator of the immune system. A complex literature exists regarding the role of IL-27 in tumor responses and the conflicting conclusions from these studies can be attributed to differences in tumor models and a lack of lineage-specific genetic tools to dissect the impact of IL-27 signaling on particular cell types. We find that IL-27 receptor alpha (WSX1)-deficient mice exhibit reduced tumor burdens following intravenous administration of B16 melanoma in comparison to wildtype mice. Further, the reduced number of macroscopic nodules corresponds with reduced tumor area within the lungs. Analysis of IL-27 reporter mice reveals a population of nonalveolar macrophage/monocytes to be the primary source of IL-27 in the lung as early as day 10 following B16 melanoma challenge. The production of IL-27 in the lung following tumor challenge and the improved resistance to intravenous dissemination of B16 melanoma by WSX1-deficient mice suggests production of IL-27 suppresses immune responses to melanoma within the lung. In line with these data, we find that tumor nodules in the lungs of WSX1-deficient mice have increased infiltration of CD3+ cells observed by histologic analysis in comparison to tumor nodules in wild-type mice. These data support a model where IL-27 suppresses the infiltration or activity of T-cells responding to metastatic melanoma and suggest that novel strategies targeting IL-27 signaling in the tumor microenvironment may synergize with current immunotherapy approaches to improve T-cell responses to tumors. Citation Format: Anthony T. Phan, Christopher A. Hunter. Defining the role of IL-27 signaling on responses to metastatic melanoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B202.

Endoplasmic Reticulum Stress Protein GRP78 Modulates Lipid Metabolism to Control Drug Sensitivity and Antitumor Immunity in Breast Cancer.

The unfolded protein response is an endoplasmic reticulum stress pathway mediated by the protein chaperone glucose regulated-protein 78 (GRP78). Metabolic analysis of breast cancer cells shows that GRP78 silencing increases the intracellular concentrations of essential polyunsaturated fats, including linoleic acid. Accumulation of fatty acids is due to an inhibition of mitochondrial fatty acid transport, resulting in a reduction of fatty acid oxidation. These data suggest a novel role of GRP78-mediating cellular metabolism. We validated the effect of GRP78-regulated metabolite changes by treating tumor-bearing mice with tamoxifen and/or linoleic acid. Tumors treated with linoleic acid plus tamoxifen exhibited reduced tumor area and tumor weight. Inhibition of either GRP78 or linoleic acid treatment increased MCP-1 serum levels, decreased CD47 expression, and increased macrophage infiltration, suggesting a novel role for GRP78 in regulating innate immunity. GRP78 control of fatty acid oxidation may represent a new homeostatic function for GRP78. Cancer Res; 76(19); 5657-70. ©2016 AACR.

Abstract 1665: Piperlongumine (PPLGM) has potent tumor-radiosensitizing properties in a mouse model of lung cancer

Abstract Background: Although over half of all lung cancer patients receive radiation therapy as part of their treatment plan, the therapeutic window of radiation is quite small, and it is difficult to deliver tumoricidal doses of radiation without the development of severe adverse effects in the nearby normal tissues. An agent that could sensitize lung cancer tissue to radiotherapy while sparing damage to normal tissues is still an unmet need. Piperlongumine (PPLGM), a natural product of the plant Piper longum has shown potent anti-carcinogenic properties through an ROS-inducing mechanism. Our hypothesis is that PPLGM will sensitize tumor cells to radiation-induced cell death in an orthotopic murine model of lung cancer. Methods: 7-week old female C57BL/6 mice were injected intravenously with 1×106 Lewis lung carcinoma (LLC) cells. Mouse cohorts (n = 30/group) were administered PPLGM (1 mg/kg of body weight) or vehicle daily by oral gavage initiated 7 days post-LLC injection. At 14 days post-LLC injection, 15 mice per treatment cohort were exposed to 12 Gy thoracic X-ray radiation treatment (XRT). Administration of PPLGM or vehicle continued 7 days post-XRT. Mice were euthanized 14 days post-XRT and evaluated for total lung weight and tumor burden using morphometric analysis. Additionally, we assessed the potential for a dual role of PPLGM (0.1-2.5μM) as a radiosensitizer of cancer cells (LLC) and a radioprotector of normal lung cells (pulmonary microvascular endothelial cells (PMVEC)) via clonogenic survival assays. Results: Using LC/MS/MS we were able to detect significantly elevated levels of PPLGM above baseline in mouse plasma and lung tissue. We observed a significant (p<0.01) increase in lung weight (from 0.14 g to 0.36 g), an indicator of tumor burden, that was significantly (p<0.01) reduced by exposure to a tumoricidal dose of XRT. Importantly, while thoracic XRT-alone reduced tumor burden by 48%, administration of PPLGM+XRT radiosensitized tumor and enhanced tumor killing by XRT, leading to a significant (p<0.05) 56% reduction in lung weight. Morphometric analysis of lung sections confirmed that PPLGM-alone significantly (p<0.01) reduced tumor area relative to vehicle, irrespective of radiation exposure. While exposure to XRT significantly (p<0.01) reduced tumor volume by 65% (from 44.4 ± 7.6% to 15.4 ± 4.5% tumor area), lung sections from mice exposed to PPLGM+XRT significantly (p<0.05) potentiated radiation-induced tumor eradication (reduction in tumor area to 5.6 ± 3.0%) above XRT-only exposure. Furthermore, PPLGM significantly (p<0.05) radiosensitized LLC dose-dependently to gamma-radiation killing, while trending towards radioprotecting normal lung cells. Conclusion: These findings provide evidence that PPLGM is a potent radiosensitizing agent that could aid in the eradication of irradiated tumors in the clinical setting. Funding: Funded in part by NIH-1R21CA178654-01 Citation Format: Anastasia Velalopoulou, Ralph A. Pietrofesa, Raymond Luke, Katie Reindl, Melpo Christofidou-Solomidou. Piperlongumine (PPLGM) has potent tumor-radiosensitizing properties in a mouse model of lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1665.

Abstract 1257: In vivo inhibition of GRP78 potentiates antiestrogen therapy effectiveness in ER+ breast tumors

Abstract Over 232,000 new cases of invasive breast cancer are diagnosed annually within the United States; 70% express the estrogen receptor-α (ER) and may be treated with therapies targeting ER, such as tamoxifen (TAM). While initially effective, over 50% of patients treated with these therapies will develop or not respond, thereby making understanding the molecular mechanism of antiestrogen resistance critical component of breast cancer biology. We have previously determined that the unfolded protein response protein chaperone, glucose regulated protein 78 (GRP78), mediates antiestrogen resistance in ER+ breast cancer cells. Mice bearing LCC1 (TAM sensitive) or LCC9 (TAM resistant) xenografts were treated with 30μM 1x weekly IP injection of human GRP78-targeting morpholino and/or a diet containing 400 ppm tamoxifen citrate for 4-6 weeks. Tumor area was measured using calipers and tumor wet weight measured at sacrifice. Combination of GRP78 knockdown and tamoxifen treatment significantly reduced LCC9 tumor area and tumor wet weight, suggesting inhibition of GRP78 restores antiestrogen sensitivity in vivo. TAM and GRP78 morpholino+TAM treatments both reduced LCC1 tumor growth, however the combination of GRP78 inhibition and TAM significantly reduced tumor area and tumor wet weight when compared to TAM treatment alone. These data suggest that inhibiting GRP78 in combination with antiestrogens is more effective for the treatment of breast cancer than single agent endocrine therapy. Metabolic analysis of LCC1 and LCC9 breast cancer cells indicate that inhibition of GRP78 through RNAi results in an increase in the intracellular concentrations of the n-3 and n-6 essential polyunsaturated fats (PUFA) linoleic and linolenic acid. We further show that the accumulation of essential fatty acids is due to an inhibition of mitochondrial fatty acid transport resulting from a reduction in SREBP1/SREBP2 transcription and CPT1A protein expression. The reduced mitochondrial fatty acid transport is also associated with an inhibition of fatty acid oxidation, suggesting a novel role of GRP78 mediating cellular metabolism. We validated the effect of the GRP78-regulated metabolites by treating mice bearing LCC9 xenografts with TAM, 0.25 mg/day linoleic acid by oral gavage, or linoleic acid+TAM for 6 weeks. LCC9 tumors treated with linoleic acid+TAM demonstrated reduced tumor area and tumor weight when compared with TAM or linoleic acid treatment alone. These data suggest that the in vivo effect of GRP78 inhibition is mediated, in part, by increased linoleic acid concentrations. Moreover, the novel role of GRP78 controlling fatty acid oxidation may represent a new homeostatic ability of GRP78 to modify fatty acid metabolism and demonstrates the importance of targeting GRP78 in breast cancer treatment. Citation Format: Katherine L. Cook, Robert Clarke. In vivo inhibition of GRP78 potentiates antiestrogen therapy effectiveness in ER+ breast tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1257. doi:10.1158/1538-7445.AM2015-1257

Open-Label, Exploratory Phase II Trial of Oral Itraconazole for the Treatment of Basal Cell Carcinoma

Itraconazole, a US Food and Drug Administration-approved antifungal drug, inhibits the Hedgehog (HH) signaling pathway, a crucial driver of basal cell carcinoma (BCC) tumorigenesis, and reduces BCC growth in mice. We assessed the effect of itraconazole on the HH pathway and on tumor size in human BCC tumors. Patients with ≥ one BCC tumor > 4 mm in diameter were enrolled onto two cohorts to receive oral itraconazole 200 mg twice per day for 1 month (cohort A) or 100 mg twice per day for an average of 2.3 months (cohort B). The primary end point was change in biomarkers: Ki67 tumor proliferation and HH activity (GLI1 mRNA). Secondary end points included change in tumor size in a subset of patients with multiple tumors. A total of 29 patients were enrolled, of whom 19 were treated with itraconazole. Itraconazole treatment was associated with two adverse events (grade 2 fatigue and grade 4 congestive heart failure). Itraconazole reduced cell proliferation by 45% (P = .04), HH pathway activity by 65% (P = .03), and reduced tumor area by 24% (95% CI, 18.2% to 30.0%). Of eight patients with multiple nonbiopsied tumors, four achieved partial response, and four had stable disease. Tumors from untreated control patients and from those previously treated with vismodegib showed no significant changes in proliferation or tumor size. Itraconazole has anti-BCC activity in humans. These results provide the basis for larger trials of longer duration to measure the clinical efficacy of itraconazole, especially relative to other HH pathway inhibitors.

O-1602, an atypical cannabinoid, inhibits tumor growth in colitis-associated colon cancer through multiple mechanisms

Cannabinoids have antiinflammatory and antitumorigenic properties. Some cannabinoids, such as O-1602, have no or only little affinity to classical cannabinoid receptors but exert cannabinoid-like antiinflammatory effects during experimental colitis. Here, we investigated whether O-1602 shows antitumorigenic effects in colon cancer cells and whether it could reduce tumorigenesis in the colon in vivo. The colon cancer cell lines HT-29 and SW480 were used to study the effect of O-1602 on viability and apoptosis. The effect of O-1602 on tumor growth in vivo was studied in a colitis-associated colon cancer mouse model. O-1602 decreased viability and induced apoptosis in colon cancer cells in a concentration-dependent manner (0.1-10 μM). In the mouse model, treatment with O-1602 (3 mg/kg, i.p., 12×) reduced tumor area by 50 % and tumor incidence by 30 %. Histological scoring revealed a significant decrease in tumor load. In tumor tissue, O-1602 decreased levels of proliferating cell nuclear antigen (PCNA), activation of oncogenic transcription factors STAT3 and NFκB p65, and expression of TNF-α while levels for proapoptotic markers, such as p53 and BAX, increased. The in vivo effects of O-1602 on PCNA, BAX, and p53 were also observed in colon cancer cells. The data provide a novel insight into antitumorigenic mechanisms of atypical cannabinoids. O-1602 exerts antitumorigenic effects by targeting colon cancer cells as well as proinflammatory pathways known to promote colitis-associated tumorigenesis. Due to its lack of central sedation, O-1602 could be an interesting compound for the treatment of colon and possibly other cancers.

Expression of NHERF1 in colonic tumors induced by 1,2-dimethylhydrazine in rats is independent of plasma ovarian steroids.

In normal embryonic fibroblasts, the Na(+)/H(+) exchanger regulator factor 1 (NHERF1) stabilizes E-cadherin/β-catenin binding and the lack of NHERF1 expression promotes cell transformation thus acting as a tumor suppressor gene. We here tested the hypothesis that NHERF1 could act as a tumor suppressor gene in colon cancer as a mediator of estrogens' protective actions in colon carcinogenesis. We studied the expression and localization of NHERF1 and β-catenin by immunohistochemistry in colonic tumors induced by 1,2 dimethylhidrazine (DMH) in Sprague-Dawley rats. One group of the rats treated with the carcinogen was ovariectomized (OVX) in the middle of the tumor induction, simulating a human menopausal condition. We observed a protective role of estrogens in colon cancer, as non-ovariectomized rats (DMH) had a reduced tumor area compared with the ovariectomized group (DMH + OVX; mean ± SE) 28.98 ± 4.65 vs. 67.58 ± 8.69 (p < 0.00380). Despite the lack of plasma estrogen stimulation, we found abundant expression of NHERF1 in colon tumors from ovariectomized rats. NHERF1 was mainly localized in the cytoplasm of the adenocarcinoma cells and lost the apical localization previously reported in normal colon tissue. We also detected expression of NHERF1 by western blot in the SW48, CACO-2, and HT29 colon cancer cell lines. Non-estrogenic factors in plasma or the tumor microenvironment may regulate NHERF1 expression in transformed colon epithelial cells. Further studies are required to understand the regulation of NHERF1 expression in colon cancer tissue.

Requirement of TGFβ Signaling for SMO-mediated Carcinogenesis

Hedgehog (Hh) signaling, via the key signal transducer Smoothened (SMO) and Gli transcription factors, is essential for embryonic development and carcinogenesis. At present, the molecular mechanism of Hh signaling-mediated carcinogenesis is not completely understood. Using a mouse model (K14cre/R26SmoM2) of SMO-mediated basal cell carcinoma development, we identified TGFβ2 as a major Hh-regulated gene. TGFβ2 expression was high in the keratinocytes, with activated TGFβ signaling (indicated by elevated expression of phosphorylated SMAD2/3) detected in both tumor and stroma. The significance of TGFβ signaling for SMO function was demonstrated in two assays. Down-regulation of TGFβ2 expression prevented Hh signaling-dependent osteoblast differentiation and motor neuron differentiation. Furthermore, inhibition of TGFβ signaling by TGFβ receptor I inhibitor SD208 significantly reduced tumor area in K14cre/R26SmoM2 mice. Tumor shrinkage in mice was associated with an increased number of lymphocytes, suggesting an immune suppression role of TGFβ signaling. The relevance of our results to human cancer is reflected by the fact that human basal cell carcinomas, which almost always harbor activated Hh signaling, have activated TGFβ signaling, as indicated by high levels of phosphorylated SMAD2 and SMAD3 in tumor and stroma. Together, our data indicate that TGFβ signaling is critical for Hh signaling-mediated carcinogenesis.

The role of NFATc1 in tumor T cell responses to lung cancer (101.6)

Abstract Nuclear factor of activated T cells (NFAT) proteins represent a Ca2+/calcineurin- regulated family of transcription factors that control T cell differentiation processes. Specifically NFATc1 and NFATc2 are highly expressed in peripheral T cells and activate the IL-2 and IL-4 promoters, suggesting that the Ca2+-regulated calcineurin/NFAT cascade controls alternative pathways of T cell activation and peripheral tolerance. NFATc1 is strongly activated upon antigen dependent T cell receptor engagement and induces genes that are involved in T cell effector function. In this study we describe that patients affected by lung Adenocarcinoma showed increased NFATc1 mRNA and protein levels in their lungs suggesting an involvement of NFATc1 on lung tumor development. To define the role of NFATc1 on lung cancer we start to analyze NFATc1 deficient mice in a murine model of lung Melanoma. Since NFATc1-deficient mice die at the embryonic stage, we bred conditional NFATc1 mice with CD4-Cre mice to gain insight into the role of NFATc1 in tumor T cell responses. Our preliminary results in this model showed a reduced tumor area in NFATc1-deficient mice compared to wild type mice. We also demonstrated here, that naïve T cells, isolated from NFATc1-deficient mice, produced less IL-17 under Th17- skewing conditions compared to those isolated from wild-type mice indicating a pathogenetic effect mediated by NFATc1 via IL-17 in lung tumor that must be further investigated.

RANK ligand inhibition plus docetaxel improves survival and reduces tumor burden in a murine model of prostate cancer bone metastasis

Tumor cells induce excessive osteoclastogenesis, mediating pathologic bone resorption and subsequent release of growth factors and calcium from bone matrix, resulting in a "vicious cycle" of bone breakdown and tumor proliferation. RANK ligand (RANKL) is an essential mediator of osteoclast formation, function, and survival. In metastatic prostate cancer models, RANKL inhibition directly prevents osteolysis via blockade of osteoclastogenesis and indirectly reduces progression of skeletal tumor burden by reducing local growth factor and calcium concentrations. Docetaxel, a well-established chemotherapy for metastatic hormone-refractory prostate cancer, arrests the cell cycle and induces apoptosis of tumor cells. Suppression of osteoclastogenesis through RANKL inhibition may enhance the effects of docetaxel on skeletal tumors. We evaluated the combination of the RANKL inhibitor osteoprotegerin-Fc (OPG-Fc) with docetaxel in a murine model of prostate cancer bone metastasis. Tumor progression, tumor area, and tumor proliferation and apoptosis were assessed. OPG-Fc alone reduced bone resorption (P < 0.001 versus PBS), inhibited progression of established osteolytic lesions, and reduced tumor area (P < 0.0001 versus PBS). Docetaxel alone reduced tumor burden (P < 0.0001 versus PBS) and delayed the development of osteolytic lesions. OPG-Fc in combination with docetaxel suppressed skeletal tumor burden (P = 0.0005) and increased median survival time by 16.7% (P = 0.0385) compared with docetaxel alone. RANKL inhibition may enhance docetaxel effects by increasing tumor cell apoptosis as evident by increased active caspase-3. These studies show that inhibition of RANKL provides an additive benefit to docetaxel treatment in a murine model of prostate cancer bone metastasis and supports clinical evaluation of this treatment option in patients.

Accelerated Bone Resorption, Due to Dietary Calcium Deficiency, Promotes Breast Cancer Tumor Growth in Bone

The skeleton is a major site of breast cancer metastases. High bone turnover increases risk of disease progression and death. However, there is no direct evidence that high bone turnover is causally associated with the establishment and progression of metastases. In this study, we investigate the effects of high bone turnover in a model of breast cancer growth in bone. Female nude mice commenced a diet containing normal (0.6%; 'Normal-Ca') or low (0.1%; 'Low-Ca') calcium content. Mice were concurrently treated with vehicle or osteoprotegerin (1 mg/kg/d s.c; n = 16 per group). Three days later (day 0), 50,000 Tx-SA cells (variant of MDA-MB-231 cells) were implanted by intratibial injection. On day 0, mice receiving Low-Ca had increased serum parathyroid hormone (PTH) and tartrate-resistant acid phosphatase 5b levels, indicating secondary hyperparathyroidism and high bone turnover, which was maintained until day 17. Osteoprotegerin increased serum PTH but profoundly reduced bone resorption. On day 17, in mice receiving Low-Ca alone, lytic lesion area, tumor area, and cancer cell proliferation increased by 43%, 24%, and 24%, respectively, compared with mice receiving Normal Ca (P < 0.01). Osteoprotegerin treatment completely inhibited lytic lesions, reduced tumor area, decreased cancer cell proliferation, and increased cancer cell apoptosis. Increased bone turnover, due to dietary calcium deficiency, promotes tumor growth in bone, independent of the action of PTH. Breast cancer patients frequently have low dietary calcium intake and high bone turnover. Treatment to correct calcium insufficiency and/or treatment with antiresorptive agents, such as osteoprotegerin, may be of benefit in the adjuvant as well as palliative setting.

Systemic administration of interferon‐beta expressing human umbilical cord matrix stem (UCMS‐ IFN‐β) cells reduce metastatic lung cancer burden in SCID mice.

Umbilical cord matrix stem (UCMS) cells are derived from Wharton’s jelly and have been shown to express genes characteristic of primitive stem cells. Stem cells that migrate specifically to tumors may allow targeted delivery of therapeutic agents that otherwise may have severe side effects. To evaluate the selective engraftment and therapeutic efficiency of human UCMS cells, MDA 231 cells were intravenously (i.v) injected into SCID mice, followed by three weekly i.v injections of UCMS cells engineered to express interferon beta (UCMS-IFN-β). To evaluate the synergistic effect of 5-Fluorouracil (5-FU) and IFN-β, MDA 231 cells were i.v injected into SCID mice, followed by three weekly i.v injections of UCMS-IFN-β cells and three weekly i.p injections of 5-FU. In both of the above experiments, mice were euthanatized one week after the last UCMS cell transplant and lung weights were compared to the controls to determine the differences in tumor burden. After transplantation of UCMS-IFN-β cells into MDA 231 tumor-bearing mice, UCMS cells were found near or within metastatic lung tumors but not in other tissues, and in these animals, the lung weight was significantly less than MDA 231 tumor-bearing animals that received saline injections. Histologically there was a significantly reduced tumor area in MDA 231 tumor bearing lungs after UCMS-IFN-β treatment. When 5-FU was given along with UCMS-IFN-β cells there was further reduction in tumor area. These results indicate the usability of UCMS cells in cancer therapy. Thus, UCMS cells can potentially be used for targeted delivery of cancer therapeutics.

Tumorigenesis disrupts hormonal regulation of tenascin expression in regressing Dunning R 3327 H prostate carcinoma.

We recently reported that androgen ablation either by orchiectomy or antiandrogen treatment resulted in the expression of the extracellular matrix glycoprotein tenascin in the regressing rat prostate. With the study presented here we investigated whether tenascin is expressed in the Dunning R 3327 H tumor and if orchiectomy and antiandrogen treatment affect tenascin expression. Experimentally, male rats were inoculated s.c. with pieces of Dunning tumor into the hind limb of both sides. Three months after inoculation rats were either orchiectomized or received a daily dose of 3 mg of cyproterone acetate or flutamide. Following a treatment period of 13 weeks, orchiectomy reduced tumor area by more than 60% compared to untreated controls. Cyproterone acetate and flutamide reduced tumor area significantly up to 30%. The amount and intensity of tenascin immunoreactivity appeared to be independent of the hormonal treatment and rather correlated to the content of tumor stroma. Those tumors with small, densely packed glandular ducts possessing almost no stromal tissue stained weakly for tenascin, whereas those tumors with larger ducts and significant stroma stained intensely. Staining intensity was particularly high at these sites where tumors infiltrated neighboring tissues, in proximity of infiltrating blood cells and close to necrotic areas. In summary, our results demonstrate a specific pattern of tenascin expression in Dunning R 3327 H rat prostate carcinomas, which appear to be independent of the hormonal treatment. We therefore conclude that tumorigenesis disrupts hormonal regulation of tenascin expression which we detected in the normal prostate gland after treatment with antiandrogens.