Relative Contribution of Cell Proliferation and Microvascularity to Metastasis in Malignant Uveal Melanoma

Uveal melanoma (UM) is the second most common primary intraocular cancer worldwide. It is a relatively rare cancer, but still the second most common type of primary malignant melanoma in humans. UM is a slowly growing tumor, and gives rise to distant metastasis mainly to the liver via the bloodstream. About 40% of patients with UM die of metastatic disease within 10 years of diagnosis, irrespective of the type of treatment. During the last decade, two main lines of research have aimed to achieve enhanced understanding of the metastasis process and accurate prognosis of patients with UM. One emphasizes the characteristics of tumor cells, particularly their nucleoli, and markers of proliferation, and the other the characteristics of tumor blood vessels. Of several morphometric measurements, the mean diameter of the ten largest nucleoli (MLN) has become the most widely applied. A large MLN has consistently been associated with high likelihood of dying from UM. Blood vessels are of paramount importance in metastasis of UM. Different extravascular matrix patterns can be seen in UM, like loops and networks. This presence is associated with death from metastatic melanoma. However, the density of microvessels is also of prognostic importance. This study was undertaken to help understanding some histopathological factors which might contribute to developing metastasis in UM patients. Factors which could be related to tumor progression to metastasis disease, namely nucleolar size, MLN, microvascular density (MVD), cell proliferation, and The Insulin-like Growth Factor 1 Receptor(IGF-1R), were investigated. The primary aim of this thesis was to study the relationship between prognostic factors such as tumor cell nucleolar size, proliferation, extravascular matrix patterns, and dissemination of UM, and to assess to what extent there is a relationship to metastasis. The secondary goal was to develop a multivariate model which includes MLN and cell proliferation in addition to MVD, and which would fit better with population-based, melanoma-related survival data than previous models. I studied 167 patients with UM, who developed metastasis even after a very long time following removal of the eye, metastatic disease was the main cause of death, as documented in the Finnish Cancer Registry and on death certificates. Using an independent population-based data set, it was confirmed that MLN and extravascular matrix loops and networks were unrelated, independent predictors of survival in UM. Also, it has been found that multivariate models including MVD in addition to MLN fitted significantly better with survival data than models which excluded MVD. This supports the idea that both the characteristics of the blood vessels and the cells are important, and the future direction would be to look for the gene expression profile, whether it is associated more with MVD or MLN. The former relates to the host response to the tumor and may not be as tightly associated with the gene expression profile, yet most likely involved in the process of hematogenous metastasis. Because fresh tumor material is needed for reliable genetic analysis, such analysis could not be performed Although noninvasive detection of certain extravascular matrix patterns is now technically possible,in managing patients with UM, this study and tumor genetics suggest that such noninvasive methods will not fully capture the process of clinical metastasis. Progress in resection and biopsy techniques is likely in the near future to result in fresh material for the ophthalmic pathologist to correlate angiographic data, histopathological characteristics such as MLN, and genetic data. This study supported the theory that tumors containing epithelioid cells grow faster and have poorer prognosis when studied by cell proliferation in UM based on Ki-67 immunoreactivity. Cell proliferation index fitted best with the survival data when combined with MVD, MLN, and presence of epithelioid cells. Analogous with the finding that high MVD in primary UM is associated with shorter time to metastasis than low MVD, high MVD in hepatic metastasis tends to be associated with shorter survival after diagnosis of metastasis. Because the liver is the main organ for metastasis from UM, growth factors largely produced in the liver hepatocyte growth factor, epidermal growth factor and insulin-like growth factor-1 (IGF-1) together with their receptors may have a role in the homing and survival of metastatic cells. Therefore the association between immunoreactivity for IGF-1R in primary UM and metastatic death was studied. It was found that immunoreactivity for IGF-IR did not independently predict metastasis from primary UM in my series.

Immunotherapeutic efficacy of Mycobacterium indicus pranii in eliciting anti-tumor T cell responses: Critical roles of IFN gamma

Mycobacterium indicus pranii (MIP) is approved for use as an adjuvant (Immuvac/Cadi-05) in the treatment of leprosy. In addition, its efficacy is being investigated in clinical trials on patients with tuberculosis and different tumors. To evaluate and delineate the mechanisms by which autoclaved MIP enhances anti-tumor responses, the growth of solid tumors consisting of Sp2/0 (myeloma) and EL4 (thymoma) cells was studied in BALB/c and C57BL/6 mice, respectively. Treatment of mice with a single intra-dermal (i.d.) injection of MIP 3 days after Sp2/0 implantation greatly suppresses tumor growth. MIP treatment of tumor bearing mice lowers Interleukin (IL)6 but increases IL12p70 and IFN? amounts in sera. Also, increase in CD8+ T cell mediated lysis of specific tumor targets and production of high amounts of IL2 and IFN? by CD4+ T cells upon stimulation with specific tumor antigens in MIP treated mice is observed. Furthermore, MIP is also effective in reducing the growth of EL4 tumors; however, this efficacy is reduced in Ifn?-/- mice. In fact, several MIP mediated anti-tumor responses are greatly abrogated in Ifn?-/- mice: increase in serum Interleukin (IL)12p70 amounts, induction of IL2 and lysis of EL4 targets by splenocytes upon stimulation with specific tumor antigens. Interestingly, tumor-induced increase in serum IL12p70 and IFN? and reduction in growth of Sp2/0 and EL4 tumors by MIP are not observed in nonobese diabetic severe combined immunodeficiency mice. Overall, our study clearly demonstrates the importance of a functional immune network, in particular endogenous CD4+ and CD8+ T cells and IFN?, in mediating the anti-tumor responses by MIP.

Expression of Sonic Hedgehog During Cell Proliferation in the Human Cerebellum

The regulation of cell proliferation in the external granular layer (EGL) of the developing cerebellum is important for its normal patterning. An important signal that regulates EGL cell proliferation is Sonic hedgehog (Shh). Shh is secreted by the Purkinje cells (PC) and has a mitogenic effect on the granule cell precursors of the EGL. Deregulation of Shh signaling has been associated with abnormal development, and been implicated in medulloblastomas, which are tumors that arise from the cerebellum. Given the importance of the Shh pathway in cerebellum development and disease, there has been no systematic study of its expression pattern during human cerebellum development. In this study, we describe the expression pattern of Shh, its receptor patched, smoothened, and its effectors that belong to the Gli family of transcription factors, during normal human cerebellum development from 10 weeks of gestational age, and in medulloblastomas that represents a case of abnormal cell proliferation in the cerebellum. This expression pattern is compared to equivalent stages in the normal development of cerebellum in mouse, as well as in tumors. Important differences between human and mouse that reflect differences in the normal developmental program between the 2 species are observed. First, in humans there appears to be a stage of Shh signaling within the EGL, when the PC are not yet the source of Shh. Second, unlike in the postnatal mouse cerebellum, expression of Shh in the PC in the postnatal human cerebellum is downregulated. Finally, medulloblastomas in the human but not in patched heterozygote mouse express Shh. These results highlight cross-species differences in the regulation of the Shh signaling pathway.

Introduction of SV40ER and hTERT into mammospheres generates breast cancer cells with stem cell properties

Emerging evidence suggests that cancers arise in stem/progenitor cells. Yet, the requirements for transformation of these primitive cells remains poorly understood. In this study, we have exploited the `mammosphere' system that selects for primitive mammary stem/progenitor cells to explore their potential and requirements for transformation. Introduction of Simian Virus 40 Early Region and hTERT into mammosphere-derived cells led to the generation of NBLE, an immortalized mammary epithelial cell line. The NBLEs largely comprised of bi-potent progenitors with long-term self-renewal and multi-lineage differentiation potential. Clonal and karyotype analyses revealed the existence of heterogeneous population within NBLEs with varied proliferation, differentiation and sphere-forming potential. Significantly, injection of NBLEs into immunocompromised mice resulted in the generation of invasive ductal adenocarcinomas. Further, these cells harbored a sub-population of CD44(+)/CD24(-) fraction that alone had sphere- and tumor-initiating potential and resembled the breast cancer stem cell gene signature. Interestingly, prolonged in vitro culturing led to their further enrichment. The NBLE cells also showed increased expression of stemness and epithelial to mesenchymal transition markers, deregulated self-renewal pathways, activated DNA-damage response and cancer-associated chromosomal aberrations-all of which are likely to have contributed to their tumorigenic transformation. Thus, unlike previous in vitro transformation studies that used adherent, more differentiated human mammary epithelial cells our study demonstrates that the mammosphere-derived, less-differentiated cells undergo tumorigenic conversion with only two genetic elements, without requiring oncogenic Ras. Moreover, the striking phenotypic and molecular resemblance of the NBLE-generated tumors with naturally arising breast adenocarcinomas supports the notion of a primitive breast cell as the origin for this subtype of breast cancer. Finally, the NBLEs represent a heterogeneous population of cells with striking plasticity, capable of differentiation, self-renewal and tumorigenicity, thus offering a unique model system to study the molecular mechanisms involved with these processes. Oncogene (2012) 31, 1896-1909; doi:10.1038/onc.2011.378; published online 29 August 2011

Oncogenic MicroRNA-155 Down-regulates Tumor Suppressor CDC73 and Promotes Oral Squamous Cell Carcinoma Cell Proliferation IMPLICATIONS FOR CANCER THERAPEUTICS

The CDC73 gene is mutationally inactivated in hereditary and sporadic parathyroid tumors. It negatively regulates beta-catenin, cyclin D1, and c-MYC. Down-regulation of CDC73 has been reported in breast, renal, and gastric carcinomas. However, the reports regarding the role of CDC73 in oral squamous cell carcinoma (OSCC) are lacking. In this study we show that CDC73 is down-regulated in a majority of OSCC samples. We further show that oncogenic microRNA-155 (miR-155) negatively regulates CDC73 expression. Our experiments show that the dramatic up-regulation of miR-155 is an exclusive mechanism for down-regulation of CDC73 in a panel of human cell lines and a subset of OSCC patient samples in the absence of loss of heterozygosity, mutations, and promoter methylation. Ectopic expression of miR-155 in HEK293 cells dramatically reduced CDC73 levels, enhanced cell viability, and decreased apoptosis. Conversely, the delivery of a miR-155 antagonist (antagomir-155) to KB cells overexpressing miR-155 resulted in increased CDC73 levels, decreased cell viability, increased apoptosis, and marked regression of xenografts in nude mice. Cotransfection of miR-155 with CDC73 in HEK293 cells abrogated its pro-oncogenic effect. Reduced cell proliferation and increased apoptosis of KB cells were dependent on the presence or absence of the 3'-UTR in CDC73. In summary, knockdown of CDC73 expression due to overexpression of miR-155 not only adds a novelty to the list of mechanisms responsible for its down-regulation in different tumors, but the restoration of CDC73 levels by the use of antagomir-155 may also have an important role in therapeutic intervention of cancers, including OSCC.

Primary Microcephaly Gene MCPH1 Shows Signatures of Tumor Suppressors and Is Regulated by miR-27a in Oral Squamous Cell Carcinoma

Mutations in the MCPH1 (microcephalin 1) gene, located at chromosome 8p23.1, result in two autosomal recessive disorders: primary microcephaly and premature chromosome condensation syndrome. MCPH1 has also been shown to be downregulated in breast, prostate and ovarian cancers, and mutated in 1/10 breast and 5/41 endometrial tumors, suggesting that it could also function as a tumor suppressor (TS) gene. To test the possibility of MCPH1 as a TS gene, we first performed LOH study in a panel of 81 matched normal oral tissues and oral squamous cell carcinoma (OSCC) samples, and observed that 14/71 (19.72%) informative samples showed LOH, a hallmark of TS genes. Three protein truncating mutations were identified in 1/15 OSCC samples and 2/5 cancer cell lines. MCPH1 was downregulated at both the transcript and protein levels in 21/41 (51.22%) and 19/25 (76%) OSCC samples respectively. A low level of MCPH1 promoter methylation was also observed in 4/40 (10%) tumor samples. We further observed that overexpression of MCPH1 decreased cellular proliferation, anchorage-independent growth in soft agar, cell invasion and tumor size in nude mice, indicating its tumor suppressive function. Using bioinformatic approaches and luciferase assay, we showed that the 3'-UTR of MCPH1 harbors two non-overlapping functional seed regions for miR-27a which negatively regulated its level. The expression level of miR-27a negatively correlated with the MCPH1 protein level in OSCC. Our study indicates for the first time that, in addition to its role in brain development, MCPH1 also functions as a tumor suppressor gene and is regulated by miR-27a.

Transcriptional Repression of Tumor Suppressor CDC73, Encoding an RNA Polymerase II Interactor, by Wilms Tumor 1 Protein (WT1) Promotes Cell Proliferation IMPLICATION FOR CANCER THERAPEUTICS

The Wilms tumor 1 gene (WT1) can either repress or induce the expression of genes. Inconsistent with its tumor suppressor role, elevated WT1 levels have been observed in leukemia and solid tumors. WT1 has also been suggested to act as an oncogene by inducing the expression of MYC and BCL-2. However, these are only the correlational studies, and no functional study has been performed to date. Consistent with its tumor suppressor role, CDC73 binds to RNA polymerase II as part of a PAF1 transcriptional regulatory complex and causes transcriptional repression of oncogenes MYC and CCND1. It also represses beta-catenin-mediated transcription. Based on the reduced level of CDC73 in oral squamous cell carcinoma (OSCC) samples in the absence of loss-of-heterozygosity, promoter methylation, and mutations, we speculated that an inhibitory transcription factor is regulating its expression. The bioinformatics analysis predicted WT1 as an inhibitory transcription factor to regulate the CDC73 level. Our results showed that overexpression of WT1 decreased CDC73 levels and promoted proliferation of OSCC cells. ChIP and EMSA results demonstrated binding of WT1 to the CDC73 promoter. The 5-azacytidine treatment of OSCC cells led to an up-regulation of WT1 with a concomitant down-regulation of CDC73, further suggesting regulation of CDC73 by WT1. Exogenous CDC73 attenuated the protumorigenic activity of WT1 by apoptosis induction. An inverse correlation between expression levels of CDC73 and WT1 was observed in OSCC samples. These observations indicated that WT1 functions as an oncogene by repressing the expression of CDC73 in OSCC. We suggest that targeting WT1 could be a therapeutic strategy for cancer, including OSCC.

A novel Monoclonal Antibody against Notch1 Targets Leukemia-associated Mutant Notch1 and Depletes Therapy Resistant Cancer Stem Cells in Solid Tumors

Higher Notch signaling is known to be associated with hematological and solid cancers. We developed a potential immunotherapeutic monoclonal antibody (MAb) specific for the Negative Regulatory Region of Notch1 (NRR). The MAb604.107 exhibited higher affinity for the ``Gain-offunction'' mutants of Notch1 NRR associated with T Acute lymphoblastic Leukemia (T-ALL). Modeling of the mutant NRR with 12 amino-acid insertion demonstrated ``opening'' resulting in exposure of the S2-cleavage site leading to activated Notch1 signaling. The MAb, at low concentrations (1-2 mu g/ml), inhibited elevated ligand-independent Notch1 signaling of NRR mutants, augmented effect of Thapsigargin, an inhibitor of mutant Notch1, but had no effect on the wild-type Notch1. The antibody decreased proliferation of the primary T-ALL cells and depleted leukemia initiating CD34/CD44 high population. At relatively high concentrations, (10-20 mu g/ml), the MAb affected Notch1 signaling in the breast and colon cancer cell lines. The Notch-high cells sorted from solid-tumor cell lines exhibited characteristics of cancer stem cells, which were inhibited by the MAb. The antibody also increased the sensitivity to Doxorubucinirubicin. Further, the MAb impeded the growth of xenografts from breast and colon cancer cells potentiated regression of the tumors along with Doxorubucin. Thus, this antibody is potential immunotherapeutic tool for different cancers.

Candida albicans Increases Tumor Cell Adhesion to Endothelial Cells In Vitro: Intraspecific Differences and Importance of the Mannose Receptor

The dimorphic fungus Candida albicans is able to trigger a cytokine-mediated pro-inflammatory response that increases tumor cell adhesion to hepatic endothelium and metastasis. To check the intraspecific differences in this effect, we used an in vitro murine model of hepatic response against C. albicans, which made clear that tumor cells adhered more to endothelium incubated with blastoconidia, both live and killed, than germ tubes. This finding was related to the higher carbohydrate/protein ratio found in blastoconidia. In fact, destruction of mannose ligand residues on the cell surface by metaperiodate treatment significantly reduced tumor cell adhesion induced. Moreover, we also noticed that the effect of clinical strains was greater than that of the reference one. This finding could not be explained by the carbohydrate/protein data, but to explain these differences between strains, we analyzed the expression level of ten genes (ADH1, APE3, IDH2, ENO1, FBA1, ILV5, PDI1, PGK1, QCR2 and TUF1) that code for the proteins identified previously in a mannoprotein-enriched pro-metastatic fraction of C. albicans. The results corroborated that their expression was higher in clinical strains than the reference one. To confirm the importance of the mannoprotein fraction, we also demonstrate that blocking the mannose receptor decreases the effect of C. albicans and its mannoproteins, inhibiting IL-18 synthesis and tumor cell adhesion increase by around 60%. These findings could be the first step towards a new treatment for solid organ cancers based on the role of the mannose receptor in C. albicans-induced tumor progression and metastasis.

Expression and Localization of Opioid Receptors in Male Germ Cells and the Implication for Mouse Spermatogenesis

The presence of endogenous opioid peptides in different testicular cell types has been extensively characterized and provides evidence for the participation of the opioid system in the regulation of testicular function. However, the exact role of the opioid system during the spermatogenesis has remained controversial since the presence of the mu-, delta-and kappa-opioid receptors in spermatogenic cells was yet to be demonstrated. Through a combination of quantitative real-time PCR, immunofluorescence, immunohistochemistry and flow cytometry approaches, we report for the first time the presence of active mu-, deltaand kappa-opioid receptors in mouse male germ cells. They show an exposition time-dependent response to opioid agonist, hence suggesting their active involvement in spermatogenesis. Our results contribute to understanding the role of the opioid receptors in the spermatogenesis and could help to develop new strategies to employ the opioid system as a biochemical tool for the diagnosis and treatment of male infertility.

Snail heterogeneity in clear cell renal cell carcinoma

Background: Intratumor heterogeneity may be responsible of the unpredictable aggressive clinical behavior that some clear cell renal cell carcinomas display. This clinical uncertainty may be caused by insufficient sampling, leaving out of histological analysis foci of high grade tumor areas. Although molecular approaches are providing important information on renal intratumor heterogeneity, a focus on this topic from the practicing pathologist' perspective is still pending. Methods: Four distant tumor areas of 40 organ-confined clear cell renal cell carcinomas were selected for histopathological and immunohistochemical evaluation. Tumor size, cell type (clear/granular), Fuhrman's grade, Staging, as well as immunostaining with Snail, ZEB1, Twist, Vimentin, E-cadherin, beta-catenin, PTEN, p-Akt, p110 alpha, and SETD2, were analyzed for intratumor heterogeneity using a classification and regression tree algorithm. Results: Cell type and Fuhrman's grade were heterogeneous in 12.5 and 60 % of the tumors, respectively. If cell type was homogeneous (clear cell) then the tumors were low-grade in 88.57 % of cases. Immunostaining heterogeneity was significant in the series and oscillated between 15 % for p110a and 80 % for Snail. When Snail immunostaining was homogeneous the tumor was histologically homogeneous in 100 % of cases. If Snail was heterogeneous, the tumor was heterogeneous in 75 % of the cases. Average tumor diameter was 4.3 cm. Tumors larger than 3.7 cm were heterogeneous for Vimentin immunostaining in 72.5 % of cases. Tumors displaying negative immunostaining for both ZEB1 and Twist were low grade in 100 % of the cases. Conclusions: Intratumor heterogeneity is a common event in clear cell renal cell carcinoma, which can be monitored by immunohistochemistry in routine practice. Snail seems to be particularly useful in the identification of intratumor heterogeneity. The suitability of current sampling protocols in renal cancer is discussed.

Hemizygosity for Atm and Brca1 influence the balance between cell transformation and apoptosis

Background: In recent years data from both mouse models and human tumors suggest that loss of one allele of genes involved in DNA repair pathways may play a central role in genomic instability and carcinogenesis. Additionally several examples in mouse models confirmed that loss of one allele of two functionally related genes may have an additive effect on tumor development. To understand some of the mechanisms involved, we examined the role of monoallelic loss or Atm and Brca1 on cell transformation and apoptosis induced by radiation. Methods: Cell transformation and apoptosis were measured in mouse embryo fibroblasts (MEF) and thymocytes respectively. Combinations of wild type and hemizygous genotypes for ATM and BRCA1 were tested in various comparisons. Results: Haploinsufficiency of either ATM or BRCA1 resulted in an increase in the incidence of radiation-induced transformation of MEF and a corresponding decrease in the proportion of thymocytes dying an apoptotic death, compared with cells from wild-type animals. Combined haploinsufficiency for both genes resulted in an even larger effect on apoptosis. Conclusions: Under stress, the efficiency and capacity for DNA repair mediated by the ATM/BRCA1 cell signalling network depends on the expression levels of both proteins.

Human uterine leiomyoma-derived fibroblasts stimulate uterine leiomyoma cell proliferation and collagen type I production, and activate RTKs and TGF beta receptor signaling in coculture.

BACKGROUND: Uterine leiomyomas (fibroids) are benign smooth muscle tumors that often contain an excessive extracellular matrix (ECM). In the present study, we investigated the interactions between human uterine leiomyoma (UtLM) cells and uterine leiomyoma-derived fibroblasts (FB), and their importance in cell growth and ECM protein production using a coculture system. RESULTS: We found enhanced cell proliferation, and elevated levels of ECM collagen type I and insulin-like growth factor-binding protein-3 after coculturing. There was also increased secretion of vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor-2, and platelet derived growth factor A and B in the media of UtLM cells cocultured with FB. Protein arrays revealed increased phosphorylated receptor tyrosine kinases (RTKs) of the above growth factor ligands, and immunoblots showed elevated levels of the RTK downstream effector, phospho-mitogen activated protein kinase 44/42 in cocultured UtLM cells. There was also increased secretion of transforming growth factor-beta 1 and 3, and immunoprecipitated transforming growth factor-beta receptor I from cocultured UtLM cells showed elevated phosphoserine expression. The downstream effectors phospho-small mothers against decapentaplegic -2 and -3 protein (SMAD) levels were also increased in cocultured UtLM cells. However, none of the above effects were seen in normal myometrial cells cocultured with FB. The soluble factors released by tumor-derived fibroblasts and/or UtLM cells, and activation of the growth factor receptors and their pathways stimulated the proliferation of UtLM cells and enhanced the production of ECM proteins. CONCLUSIONS: These data support the importance of interactions between fibroid tumor cells and ECM fibroblasts in vivo, and the role of growth factors, and ECM proteins in the pathogenesis of uterine fibroids.

Boosting high-intensity focused ultrasound-induced anti-tumor immunity using a sparse-scan strategy that can more effectively promote dendritic cell maturation.

BACKGROUND: The conventional treatment protocol in high-intensity focused ultrasound (HIFU) therapy utilizes a dense-scan strategy to produce closely packed thermal lesions aiming at eradicating as much tumor mass as possible. However, this strategy is not most effective in terms of inducing a systemic anti-tumor immunity so that it cannot provide efficient micro-metastatic control and long-term tumor resistance. We have previously provided evidence that HIFU may enhance systemic anti-tumor immunity by in situ activation of dendritic cells (DCs) inside HIFU-treated tumor tissue. The present study was conducted to test the feasibility of a sparse-scan strategy to boost HIFU-induced anti-tumor immune response by more effectively promoting DC maturation. METHODS: An experimental HIFU system was set up to perform tumor ablation experiments in subcutaneous implanted MC-38 and B16 tumor with dense- or sparse-scan strategy to produce closely-packed or separated thermal lesions. DCs infiltration into HIFU-treated tumor tissues was detected by immunohistochemistry and flow cytometry. DCs maturation was evaluated by IL-12/IL-10 production and CD80/CD86 expression after co-culture with tumor cells treated with different HIFU. HIFU-induced anti-tumor immune response was evaluated by detecting growth-retarding effects on distant re-challenged tumor and tumor-specific IFN-gamma-secreting cells in HIFU-treated mice. RESULTS: HIFU exposure raised temperature up to 80 degrees centigrade at beam focus within 4 s in experimental tumors and led to formation of a well-defined thermal lesion. The infiltrated DCs were recruited to the periphery of lesion, where the peak temperature was only 55 degrees centigrade during HIFU exposure. Tumor cells heated to 55 degrees centigrade in 4-s HIFU exposure were more effective to stimulate co-cultured DCs to mature. Sparse-scan HIFU, which can reserve 55 degrees-heated tumor cells surrounding the separated lesions, elicited an enhanced anti-tumor immune response than dense-scan HIFU, while their suppressive effects on the treated primary tumor were maintained at the same level. Flow cytometry analysis showed that sparse-scan HIFU was more effective than dense-scan HIFU in enhancing DC infiltration into tumor tissues and promoting their maturation in situ. CONCLUSION: Optimizing scan strategy is a feasible way to boost HIFU-induced anti-tumor immunity by more effectively promoting DC maturation.

Differential Apaf-1 levels allow cytochrome c to induce apoptosis in brain tumors but not in normal neural tissues.

Brain tumors are typically resistant to conventional chemotherapeutics, most of which initiate apoptosis upstream of mitochondrial cytochrome c release. In this study, we demonstrate that directly activating apoptosis downstream of the mitochondria, with cytosolic cytochrome c, kills brain tumor cells but not normal brain tissue. Specifically, cytosolic cytochrome c is sufficient to induce apoptosis in glioblastoma and medulloblastoma cell lines. In contrast, primary neurons from the cerebellum and cortex are remarkably resistant to cytosolic cytochrome c. Importantly, tumor tissue from mouse models of both high-grade astrocytoma and medulloblastoma display hypersensitivity to cytochrome c when compared with surrounding brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in the tumor tissue compared with low Apaf-1 levels in the adjacent brain tissue. These differences in Apaf-1 abundance correlate with differences in the levels of E2F1, a previously identified activator of Apaf-1 transcription. ChIP assays reveal that E2F1 binds the Apaf-1 promoter specifically in tumor tissue, suggesting that E2F1 contributes to the expression of Apaf-1 in brain tumors. Together, these results demonstrate an unexpected sensitivity of brain tumors to postmitochondrial induction of apoptosis. Moreover, they raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancer cells while sparing the surrounding brain parenchyma.