Sodium accumulation in breast cancer predicts malignancy and treatment response.

BACKGROUND: Breast cancer remains a leading cause of death in women and novel imaging biomarkers are urgently required. Here, we demonstrate the diagnostic and treatment-monitoring potential of non-invasive sodium (23Na) MRI in preclinical models of breast cancer. METHODS: Female Rag2-/- Il2rg-/- and Balb/c mice bearing orthotopic breast tumours (MDA-MB-231, EMT6 and 4T1) underwent MRI as part of a randomised, controlled, interventional study. Tumour biology was probed using ex vivo fluorescence microscopy and electrophysiology. RESULTS: 23Na MRI revealed elevated sodium concentration ([Na+]) in tumours vs non-tumour regions. Complementary proton-based diffusion-weighted imaging (DWI) linked elevated tumour [Na+] to increased cellularity. Combining 23Na MRI and DWI measurements enabled superior classification accuracy of tumour vs non-tumour regions compared with either parameter alone. Ex vivo assessment of isolated tumour slices confirmed elevated intracellular [Na+] ([Na+]i); extracellular [Na+] ([Na+]e) remained unchanged. Treatment with specific inward Na+ conductance inhibitors (cariporide, eslicarbazepine acetate) did not affect tumour [Na+]. Nonetheless, effective treatment with docetaxel reduced tumour [Na+], whereas DWI measures were unchanged. CONCLUSIONS: Orthotopic breast cancer models exhibit elevated tumour [Na+] that is driven by aberrantly elevated [Na+]i. Moreover, 23Na MRI enhances the diagnostic capability of DWI and represents a novel, non-invasive biomarker of treatment response with superior sensitivity compared to DWI alone.

Spontaneous development of Epstein-Barr Virus associated human lymphomas in a prostate cancer xenograft program.

Prostate cancer research is hampered by the lack of in vivo preclinical models that accurately reflect patient tumour biology and the clinical heterogeneity of human prostate cancer. To overcome these limitations we propagated and characterised a new collection of patient-derived prostate cancer xenografts. Tumour fragments from 147 unsupervised, surgical prostate samples were implanted subcutaneously into immunodeficient Rag2-/-γC-/- mice within 24 hours of surgery. Histologic and molecular characterisation of xenografts was compared with patient characteristics, including androgen-deprivation therapy, and exome sequencing. Xenografts were established from 47 of 147 (32%) implanted primary prostate cancers. Only 14% passaged successfully resulting in 20 stable lines; derived from 20 independent patient samples. Surprisingly, only three of the 20 lines (15%) were confirmed as prostate cancer; one line comprised of mouse stroma, and 16 were verified as human donor-derived lymphoid neoplasms. PCR for Epstein-Barr Virus (EBV) nuclear antigen, together with exome sequencing revealed that the lymphomas were exclusively EBV-associated. Genomic analysis determined that 14 of the 16 EBV+ lines had unique monoclonal or oligoclonal immunoglobulin heavy chain gene rearrangements, confirming their B-cell origin. We conclude that the generation of xenografts from tumour fragments can commonly result in B-cell lymphoma from patients carrying latent EBV. We recommend routine screening, of primary outgrowths, for latent EBV to avoid this phenomenon.

Enhanced cross-priming of naive CD8+ T cells by dendritic cells treated by the IMiDs® immunomodulatory compounds lenalidomide and pomalidomide.

The IMiDs(®) immunomodulatory compounds lenalidomide and pomalidomide are agents with anti-inflammatory, immunomodulatory and anti-cancer activity. An excellent success rate has been shown for multiple myeloma in phase I/II clinical trials leading to Food and Drug Administration approval of lenalidomide. One mechanism by which these drugs could enhance anti-tumour immunity may be through enhanced dendritic cell (DC) function. Thalidomide, a compound structurally related to lenalidomide and pomalidomide, is known to enhance DC function, and we have investigated whether its analogues, pomalidomide and lenalidomide, also have functional effects on DCs. We used mouse bone marrow-derived DCs treated with 5 or 10 µm pomalidomide, or lenalidomide from day 1 of culture. Treatment with IMiD(®) immunomodulatory compounds increased expression of Class I (H2-Kb), CD86, and pomalidomide also increased Class II (I-Ab) expression in bone marrow-derived DCs, as measured by flow cytometry. Fluorescent bead uptake was increased by up to 45% when DCs were treated with 5 or 10 µm pomalidomide or lenalidomide compared with non-treated DCs. Antigen presentation assays using DCs primed with ovalbumin, and syngeneic T cells from transgenic OTI and OTII mice (containing MHC restricted, ovalbumin-specific, T cells) showed that both pomalidomide and lenalidomide effectively increased CD8(+) T-cell cross-priming (by up to 47%) and that pomalidomide alone was effective in increasing CD4(+) T-cell priming (by 30%). Our observations suggest that pomalidomide and lenalidomide enhance tumour antigen uptake by DCs with an increased efficacy of antigen presentation, indicating a possible use of these drugs in DC vaccine therapies.

Regulation of the stem cell marker CD133 is independent of promoter hypermethylation in human epithelial differentiation and cancer.

BACKGROUND: Epigenetic control is essential for maintenance of tissue hierarchy and correct differentiation. In cancer, this hierarchical structure is altered and epigenetic control deregulated, but the relationship between these two phenomena is still unclear. CD133 is a marker for adult stem cells in various tissues and tumour types. Stem cell specificity is maintained by tight regulation of CD133 expression at both transcriptional and post-translational levels. In this study we investigated the role of epigenetic regulation of CD133 in epithelial differentiation and cancer. METHODS: DNA methylation analysis of the CD133 promoter was done by pyrosequencing and methylation specific PCR; qRT-PCR was used to measure CD133 expression and chromatin structure was determined by ChIP. Cells were treated with DNA demethylating agents and HDAC inhibitors. All the experiments were carried out in both cell lines and primary samples. RESULTS: We found that CD133 expression is repressed by DNA methylation in the majority of prostate epithelial cell lines examined, where the promoter is heavily CpG hypermethylated, whereas in primary prostate cancer and benign prostatic hyperplasia, low levels of DNA methylation, accompanied by low levels of mRNA, were found. Moreover, differential methylation of CD133 was absent from both benign or malignant CD133+/α2ß1integrinhi prostate (stem) cells, when compared to CD133-/α2ß1integrinhi (transit amplifying) cells or CD133-/α2ß1integrinlow (basal committed) cells, selected from primary epithelial cultures. Condensed chromatin was associated with CD133 downregulation in all of the cell lines, and treatment with HDAC inhibitors resulted in CD133 re-expression in both cell lines and primary samples. CONCLUSIONS: CD133 is tightly regulated by DNA methylation only in cell lines, where promoter methylation and gene expression inversely correlate. This highlights the crucial choice of cell model systems when studying epigenetic control in cancer biology and stem cell biology. Significantly, in both benign and malignant prostate primary tissues, regulation of CD133 is independent of DNA methylation, but is under the dynamic control of chromatin condensation. This indicates that CD133 expression is not altered in prostate cancer and it is consistent with an important role for CD133 in the maintenance of the hierarchical cell differentiation patterns in cancer.

The anti-cancer agents lenalidomide and pomalidomide inhibit the proliferation and function of T regulatory cells.

Lenalidomide (Revlimid; CC-5013) and pomalidomide (CC-4047) are IMiDs proprietary drugs having immunomodulatory properties that have both shown activity in cancer clinical trials; lenalidomide is approved in the United States for a subset of MDS patients and for treatment of patients with multiple myeloma when used in combination with dexamethasone. These drugs exhibit a range of interesting clinical properties, including anti-angiogenic, anti-proliferative, and pro-erythropoietic activities although exact cellular target(s) remain unclear. Also, anti-inflammatory effects on LPS-stimulated monocytes (TNF-alpha is decreased) and costimulatory effects on anti-CD3 stimulated T cells, (enhanced T cell proliferation and proinflammatory cytokine production) are observed. These drugs also cause augmentation of NK-cell cytotoxic activity against tumour-cell targets. Having shown that pomalidomide confers T cell-dependent adjuvant-like protection in a preclinical whole tumour-cell vaccine-model, we now show that lenalidomide and pomalidomide strongly inhibit T-regulatory cell proliferation and suppressor-function. Both drugs inhibit IL-2-mediated generation of FOXP3 positive CTLA-4 positive CD25high CD4+ T regulatory cells from PBMCs by upto 50%. Furthermore, suppressor function of pre-treated T regulatory cells against autologous responder-cells is abolished or markedly inhibited without drug related cytotoxicity. Also, Balb/C mice exhibit 25% reduction of lymph-node T regulatory cells after pomalidomide treatment. Inhibition of T regulatory cell function was not due to changes in TGF-beta or IL-10 production but was associated with decreased T regulatory cell FOXP3 expression. In conclusion, our data provide one explanation for adjuvant properties of lenalidomide and pomalidomide and suggest that they may help overcome an important barrier to tumour-specific immunity in cancer patients.

A novel murine model of allogeneic vaccination against renal cancer.

OBJECTIVES: To develop a murine model for whole-cell allogeneic vaccination in renal cancer, as such vaccines aim to direct immune responses against patient tumour cells, due to shared antigens between the vaccine and tumour cells. MATERIALS AND METHODS: A novel murine renal cell line, allogeneic to BALB/c, was developed from a C57BL/6 mouse by primary cell culture (RVIK). It was immortalized by HPV16 E6/E7 and transfected with ras in an attempt to improve its immunogenicity. The cell line was characterized and tested as a vaccine in a BALB/c tumour-protection model after subsequent tumour challenge with autologous RenCa tumour cells. RESULTS: RVIK alone, with no ras induced cross-reactive immunity, providing a valid non-tumorigenic allogeneic whole-cell vaccine model for renal cancer. Ras transfection per se did not improve RVIK immunity. CONCLUSIONS: RVIK is a novel immunogenic murine renal epithelial cell line, which confers protection when used as an allogeneic vaccine. It provides proof of principle for the effectiveness of allogeneic whole-cell vaccines and may therefore form the basis of a useful model of allogeneic vaccination to further optimize vaccination schedules, formulation and adjuvants for a clinical setting.

The biological effects of syngeneic and allogeneic cytokine-expressing prophylactic whole cell vaccines and the influence of irradiation in a murine melanoma model.

Allogeneic whole tumour cell vaccines are inherently practical compared with autologous vaccines. Cell lines are derived from allogeneic tumour, grown in bulk and then administered as a vaccine to the patient, following irradiation, which not only prevents any replication but also enhances antigen presentation. Protection is believed to occur through the presentation of antigens shared between the syngeneic and allogeneic tumours. Although cytokine-transfected tumour whole cell vaccines have been used clinically, little data is available comparing the effects of immunomodulatory cytokine-transfection directly on the same cells when used as both an allogeneic and autologous vaccine. To address this, weakly immunogenic B16-F10 (H-2b) murine melanoma was transfected to secrete either GM-CSF, IL-4 or IL-7. Prophylactic vaccination of both syngeneic C57/BL6 (H-2b) (B6) and allogeneic C3H/Hej (H-2k) (C3H) mice showed the effects of transfected cytokine varied between models. Both GM-CSF and IL-7 significantly (P<0.05) increased the levels of protection within syngeneic B6 mice, but had a diminished effect (P>0.05) within C3H allogeneic mice. Allogeneic B16-F10 cells and syngeneic K1735 cells generated CTL against K1735 suggesting cross-reactive immunity. Using cells labeled with fluorescent dye we demonstrate that irradiated vaccines, of either syngeneic or allogeneic origin, appear to generate potent immune responses and fragments of either vaccine remain at the injection site for up to 9 days. This study shows that protection can be enhanced in vivo by using transfected cytokine, but suggests that irradiated whole cell vaccines, of either tissue-type, are rapidly processed. This leads to the conclusion that the cytokine effects are transient and thus transfection with cytokine may be of limited long-term use in situ.

Thalidomide-derived immunomodulatory drugs as therapeutic agents.

Thalidomide, a drug originally used to treat morning sickness, was removed from the market place in the early 1960s after it was found to cause serious congenital birth defects. However, thalidomide has recently been investigated in a new light following its activity in a number of chronic diseases. Moreover, like thalidomide itself, its second-generation immunomodulatory drug (IMiD) analogues have been shown to act as powerful anticancer agents and are clearly active in the treatment of patients with relapsed multiple myeloma. These new drugs, in particular the second-generation IMiDs, lenalidomide (CC-5013, REVLIMID; Celgene Corp., NJ, USA) and CC-4047 (ACTIMID; Celgene Corp.), offer improvements over thalidomide (a first-generation IMiD) in terms of efficacy and safety in human studies. The key to the therapeutic potential of IMiDs lies in the fact that the drugs have multiple mechanisms of action, which may produce both anti-inflammatory and antitumour effects. These effects are probably contextual, depending both on the cell type and the stimulus involved. Mechanisms associated with IMiD activity include TNF-alpha-inhibitory, T cell costimulatory and antiangiogenic activities. Studies of the mechanisms of action of these drugs are ongoing and will facilitate the continued development of this class of compound in a number of diseases.