Prevent Tumor Relapse Research Articles

Myeloid STAT3 promotes formation of colitis-associated colorectal cancer in mice

Myeloid cells lacking STAT3 promote antitumor responses of NK and T cells but it is unknown if this crosstalk affects development of autochthonous tumors. We deleted STAT3 in murine myeloid cells (STAT3Δm) and examined the effect on the development of autochthonous colorectal cancers (CRCs). Formation of Azoxymethane/Dextransulfate (AOM/DSS)-induced CRCs was strongly suppressed in STAT3Δm mice. Gene expression profiling showed strong activation of T cells in the stroma of STAT3Δm CRCs. Moreover, STAT3Δm host mice were better able to control the growth of transplanted MC38 colorectal tumor cells which are known to be killed in a T cell-dependent manner. These data suggest that myeloid cells lacking STAT3 control formation of CRCs mainly via cross activation of T cells. Interestingly, the few CRCs that formed in STAT3Δm mice displayed enhanced stromalization but appeared normal in size indicating that they have acquired ways to escape enhanced tumor surveillance. We found that CRCs in STAT3Δm mice consistently activate STAT3 signaling which is implicated in immune evasion and might be a target to prevent tumor relapse.

Tenascin-C: Exploitation and collateral damage in cancer management

Despite an increasing knowledge about the causes of cancer, this disease is difficult to cure and still causes far too high a death rate. Based on advances in our understanding of disease pathogenesis, novel treatment concepts, including targeting the tumor microenvironment, have been developed and are being combined with established treatment regimens such as surgical removal and radiotherapy. Yet it is obvious that we need additional strategies to prevent tumor relapse and metastasis. Given its exceptional high expression in most cancers with low abundance in normal tissues, tenascin-C appears an ideal candidate for tumor treatment. Here, we will summarize the current applications of targeting tenascin-C as a treatment for different tumors, and highlight the potential of this therapeutic approach.

Therapies targeting cancer stem cells: Current trends and future challenges.

Traditional therapies against cancer, chemo- and radiotherapy, have multiple limitations that lead to treatment failure and cancer recurrence. These limitations are related to systemic and local toxicity, while treatment failure and cancer relapse are due to drug resistance and self-renewal, properties of a small population of tumor cells called cancer stem cells (CSCs). These cells are involved in cancer initiation, maintenance, metastasis and recurrence. Therefore, in order to develop efficient treatments that can induce a long-lasting clinical response preventing tumor relapse it is important to develop drugs that can specifically target and eliminate CSCs. Recent identification of surface markers and understanding of molecular feature associated with CSC phenotype helped with the design of effective treatments. In this review we discuss targeting surface biomarkers, signaling pathways that regulate CSCs self-renewal and differentiation, drug-efflux pumps involved in apoptosis resistance, microenvironmental signals that sustain CSCs growth, manipulation of miRNA expression, and induction of CSCs apoptosis and differentiation, with specific aim to hamper CSCs regeneration and cancer relapse. Some of these agents are under evaluation in preclinical and clinical studies, most of them for using in combination with traditional therapies. The combined therapy using conventional anticancer drugs with CSCs-targeting agents, may offer a promising strategy for management and eradication of different types of cancers.

System Models, Assays and Endpoint Parameters to Evaluate Anticancer Compounds During Preclinical Screening

The effectiveness of anticancer therapies relies on the ability of these substances to selectively eliminate the malignant cells with little or no toxicity to normal cells. The isolation in most human tumors of a rare subpopulation of cancer stem cells (CSCs) associated with chemo resistance leads to the "stem cell theory" (SCT). The SCT proposed that eliminating this fraction will eventually cure cancer but experimental data supporting this classical view are controversial and now being gradually replaced by other models. These novel models of cancer biology predict that to cure cancer only drugs or combination of drugs that eliminate all (CSCs and non-CSCs) cancer cells at once ("pankiller drugs") will be effective. The search for "pankiller drugs" will require tests to assess (i) the elimination of all cancer cells in in vitro systems (ii) the ability to eradicate the tumors and prevent tumor relapse in in vivo systems. However, at present, most drugs are being tested in assays that can only provide a picture of the short term activity of anticancer compounds. This in part explains why only a small fraction of the drugs that enter clinical trials are actually approved for clinical use. This article will provide a concise review of the systems, assays and endpoint parameters routinely used to screen for potential anticancer drugs and propose, based in the current knowledge of cancer biology, a more rationale anticancer drug screening program.

Coencapsulation of epirubicin and metformin in PEGylated liposomes inhibits the recurrence of murine sarcoma S180 existing CD133+ cancer stem-like cells

Cancer stem cells (CSCs), also known as tumor-initiating cells, which constitute a subpopulation of tumor cells, are key drivers of tumorigenesis and potential recurrence of cancer. The CSC theory has brought new opportunities as well as challenges to the development of sophisticated drug delivery systems for treating cancer. In the present study, CD133+ cells were sorted from S180 cell lines by magnetic activated cell sorting and a fraction (approximately 1.01%) of CD133+ cells with higher proliferative potential and stronger tumorigenicity in vivo compared with CD133− cells was identified. Furthermore, a procedure for the coencapsulation of epirubicin (EPI) and metformin (MET) was developed with the primary goal of eradicating the bulk population of CD133− cells and the rare population of CD133+ cancer stem-like cells, thus ultimately preventing tumor relapse. The inhibitory effect of free MET was more potent in CD133+ cells than in CD133− cells; in addition, EPI- and MET-coencapsulated liposomes exhibited increased cytotoxicity against CD133+ cells compared with liposomal EPI alone. Meanwhile, tumors in KM mice were completely eliminated upon multiple intravenous injections of liposomal EPI and MET, and tumors virtually eliminated in the experimental period, which could be attributed to the arrest of CD133+ cells in the G0/G1 phase. The coencapsulation of an anti-CSC agent with conventional chemotherapy drugs in liposomes may be a promising drug delivery strategy for fighting cancer and eradicating tumor stem cells.

CoCl2, a mimic of hypoxia, induces formation of polyploid giant cells with stem characteristics in colon cancer.

The induction of polyploidy is considered the reproductive end of cells, but there is evidence that polyploid giant cancer cells (PGCCs) contribute to cell repopulation during tumor relapse. However, the role of these cells in the development, progression and response to therapy in colon cancer remains undefined. Therefore, the main objective of this study was to investigate the generation of PGCCs in colon cancer cells and identify mechanisms of formation. Treatment of HCT-116 and Caco-2 colon cancer cells with the hypoxia mimic CoCl2 induced the formation of cells with larger cell and nuclear size (PGCCs), while the cells with normal morphology were selectively eliminated. Cytometric analysis showed that CoCl2 treatment induced G2 cell cycle arrest and the generation of a polyploid cell subpopulation with increased cellular DNA content. Polyploidy of hypoxia-induced PGCCs was confirmed by FISH analysis. Furthermore, CoCl2 treatment effectively induced the stabilization of HIF-1α, the differential expression of a truncated form of p53 (p47) and decreased levels of cyclin D1, indicating molecular mechanisms associated with cell cycle arrest at G2. Generation of PGCCs also contributed to expansion of a cell subpopulation with cancer stem cells (CSCs) characteristics, as indicated by colonosphere formation assays, and enhanced chemoresistance to 5-fluorouracil and oxaliplatin. In conclusion, the pharmacological induction of hypoxia in colon cancer cells causes the formation of PGCCs, the expansion of a cell subpopulation with CSC characteristics and chemoresistance. The molecular mechanisms involved, including the stabilization of HIF-1 α, the involvement of p53/p47 isoform and cell cycle arrest at G2, suggest novel targets to prevent tumor relapse and treatment failure in colon cancer.

Ovarian Cancer Biology and Immunotherapy

Ovarian cancer is the most lethal malignancy of the female reproductive system and the fifth leading cause of cancer death in women. In the year 2012 alone, United States had 22,280 new ovarian cancer cases and 15,500 deaths were reported. About 7%–10% of ovarian cancers result from an inherited tendency to develop the disease. Ovarian cancer has the ability to escape the immune system because of its pathological interactions between cancer cells and host immune cells in the tumor microenvironment create an immunosuppressive network that promotes tumor growth, protects the tumor from immune system. The levels of immune suppressive elements like regulatory T cells, plasmacytoid dendritic cells and cytokines such as IL-10, IL-6, TNF-α, and TGF-β are elevated in the tumor microenvironment. Vascular endothelial growth factor is known to have an immune suppressing role besides its angiogenic role in the tumor microenvironment. Ovarian cancer is associated with high mortality partly due to difficulties in early diagnosis and development of metastases. These problems may overcome by developing accurate mouse models that should mimic the complexity of human ovarian cancer. Such animal models are better suited to understand pathophysiology, metastases, and also for preclinical testing of targeted molecular therapeutics. Immunotherapy is an area of active investigation and off late many clinical trials is ongoing to prevent disease progression. The main aim of dendritic cells vaccination is to stimulate tumor specific effector T cells that can reduce tumor size and induce immunological memory to prevent tumor relapse.

Effect of adoptive T-cell therapy and intratumoral heat-killed bacteria on large tumors.

11112 Background: Preventing tumor relapse as antigen-loss variants remains a challenge in T cell therapy. T cells eradicate tumors by secreting cytokines that mediate bystander killing of tumor st...

Research perspectives: gold nanoparticles in cancer theranostics.

High recurrence rates after surgical resection remain a formidable challenge in many cancers. Although chemo- and/or radiotherapy are often applied following surgery to prevent tumor relapse, these treatments are generally accompanied by serious side effects and challenges in their delivery that limit their effectiveness. Gold nanoparticles (AuNPs), which possess unique physicochemical properties, have the potential to enhance the efficacy of these conventional treatment modalities. In this review, we briefly describe the current state of AuNP research in the area of cancer theranostics. Recent studies have investigated AuNPs' use as photothermal converters, drug carriers, radiosensitizers, and imaging probes in a wide range of applications for cancer diagnosis and therapy. AuNPs have promise in minimally invasive thermal ablation therapy, diagnostic imaging, intraoperative tumor margin delineation, and multimodal anticancer therapy. The successful translation of AuNPs into the clinic will have significant impact on the care of cancer patients using image-guided, minimally invasive approaches.

Administration Of Anti-CD20 Mab Is Highly Effective In Preventing But Ineffective In Treating Chronic GVHD While Preserving A Strong GVL Effect

Donor T cell-mediated graft versus leukemia/lymphoma (GVL) effect plays a critical role in preventing tumor relapse in leukemia/lymphoma patients treated with allogeneic hematopoietic cell transplantation (HCT). However, these same donor T cells also induce acute and chronic graft versus host disease (GVHD), which remains a major obstacle for allogeneic HCT as a curative therapy for hematological malignancies. Chronic GVHD is a systemic lupus- and scleroderma-like autoimmune syndrome. We and others reported that donor B cells play important roles in augmenting pathogenesis of chronic GVHD in mouse models and humans. Rituxan (an anti-CD20 mAb) has been used in the clinic for the treatment of ongoing chronic GVHD, but the effect is variable and minimum in some reports, and the mechanisms for this ineffectiveness remains unclear. In the current studies, we evaluated the effect of in vivo administration of a depleting anti-CD20 mAb in preventing and treating autoimmune-like chronic GVHD as well as the impact on GVL effect. With the mouse chronic GVHD model of DBA/2 donor to MHC-matched BALB/c recipient established in our lab (Blood, 2006, J. Immunol 2012), we found that one intravenous injection of anti-CD20 (50mg/kg) immediately after HCT effectively prevented induction of autoimmune-like chronic GVHD. While all (12/12) recipients treated with control IgG developed chronic GVHD with proteinuria and hair-loss and died by 30 days, none (0/12) of the anti-CD20-treated recipients developed proteinuria or hair-loss and all survived for more than 50 days after HCT without signs of chronic GVHD. In addition, the anti-CD20-treated recipients eliminated BCL1 leukemia/lymphoma cells without signs of chronic GVHD. The preventative anti-CD20 treatment had little impact on donor CD8+ T cell activation and expansion in the periphery and allowed for the strong CD8+ T cell-mediated GVL effect. The effective prevention of chronic GVHD by anti-CD20 was associated with significant changes in donor B and CD4+ T cells as well as associated with protection of host thymus. Preventive anti-CD20 treatment depleted IL-6-producing donor B cells and increased IL-10-producing B cells. In addition, the treatment also significantly reduced donor CD4+ T cell expansion, the percentage of IFN-g-producing CD4+ T as well as CD4+ T cells expressing CD8αα, the latter of which has recently been reported to represent over-activated pathogenic CD4+ T cells, such that the host thymus was protected from donor T-cell-mediated damage. Anti-CD20 or control IgG treatment of recipients with ongoing chronic GVHD 15-20 days after HCT did not show any difference in disease progress, and all (8/8) in each group died within 30 days after HCT. The ineffectiveness of anti-CD20 therapy was associated with little reduction of CD19+ B or CD4+ T cells, as the majority of CD19+ B cells became CD20- in the recipients with ongoing chronic GVHD. These results indication that anti-CD20 can effectively prevent induction of autoimmune-like chronic GVHD while preserving the GVL effect, but anti-CD20 is ineffective in treating ongoing chronic GVHD. (This study is supported by the Nesvig Lymphoma Foundation). Disclosures:Chan:Genentech Inc.: Employment.

Idiotypic Vaccination: Still a Unique form of Cancer Immunotherapy for Follicular Lymphoma after 20 Years

Idiotypic vaccination for folicular lymphoma induces a tumor-specific immune response which may kill tumor cells in vivo and prevent tumor relapse in patients. However, being based on a personalized vaccine against a person’s own tumor, large scale randomized studies have produced contradictory results. The objective of this review is to define what an idiotype is and to outline the major results of twenty years of clinical research on idiotypic vaccination. We first identified the major proofs of principle obtained by its use between 1992 and 2006, focusing on both our and others’ contributions. Then, we analyzed the results of randomized clinical trials, which have become available ove the last five years, and provided some of our most recent and original data. A combination of immunological methods should be employed for proper interpretation of immune response following idiotypic vaccination. We describe some of the methods used to measure immune responses and identify tumor idiotype sequences. Some of the older methods (e.g. ELISA) may, in some instances, be misleading, and should be validated using different methods (e.g. flow cytometry). Moreover, patients who relapse years after the end of the vaccination schedule, may have undergone changes in tumor idiotype tumor idiotypes that have undergone substantial changes and is advisable to test the tumor clone at relapse to ascertain it. While idiotypic vaccination has provided proof-of-principle of effectiveness, the data necessary for regulatory approval has yet to be generated. Therefore, better designed, confirmatory clinical trials of idiotypic vaccination are necessary.

CIK cells from recurrent or refractory AML patients can be efficiently expanded in vitro and used for reduction of leukemic blasts in vivo

Autologous cytokine-induced killer (CIK) cell transfusion may prevent tumor relapse in acute myeloid leukemia (AML). This study investigated whether CIK cells from recurrent or refractory AML patients with high peripheral leukemia cell burdens could be expanded to a clinically usable number, and it further evaluated the antitumor potentials invitro and invivo. The numbers and phenotypes of CIK cells expanded from nine AML patients and 10 healthy donors were compared. Cytotoxicity (against K562 and U937 cell lines) and cytokine secretion (interleukin-2, interferon-γ, tumor necrosis factor-α and vascular endothelial growth factor) were tested for AML-derived and healthy donor-derived CIK cells and fresh peripheral blood mononuclear cells from healthy donors. Importantly, we assessed the therapeutic effects of autologous CIK cell infusions in two patients with AML. The proportions of CD3(+)and CD3(+)CD56(+) CIK cells from patients with AML were similar to those from healthy donors, and the number of CD3(+)CD56(+) cells in AML-derived CIK cells was expanded approximately 1,020-fold. Phenotype analyses with flow cytometry showed that the leukemic cells were gradually eliminated during the process of CIK cell preparation to an almost undetectable level. Although the cytotoxic effect of AML-derived CIK cells was equivalent to that of healthy donors, AML-derived CIK cells had a significantly higher cytokine-secreting capacity. In clinical treatment, the leukemia burden in the peripheral blood of one patient was dramatically decreased after four transfusions within 4 months. CIK cells can be efficiently expanded invitro from patients with recurrent or refractory AML and may be used for reduction of leukemic blasts invivo.

Abstract 5247: Use of two markers of hypoxia to study migration, re-oxygenation and repopulation of originally hypoxic cells in MCF-7 tumor xenografts following chemotherapy

Abstract Background: Hypoxia occurs in solid tumors: chronic diffusion-limited hypoxia occurs distal from functional blood vessels, while acute hypoxia may occur because of interruptions in blood flow. Well-nourished tumor cells close to blood vessels tend to be rapidly proliferating but hypoxic cells located farther away are slowly-proliferating. Chemotherapy may spare hypoxic cells because of poor drug distribution to them and because most drugs are selectively toxic to proliferating cells. Intervals between administration of chemotherapy allow for recovery of normal tissues (e.g. repopulation of bone marrow) but might allow re-oxygenation and resumed proliferation of formerly hypoxic cells due to better supply of nutrients to them, as is observed during radiotherapy. Therefore, it is important to establish a technique whereby hypoxia can be studied allowing for the assessment of different hypoxia-modulating therapies that can be used to prevent tumor relapse. Methods: Two specific markers of hypoxic cells (pimonidazole [pimo] and EF5) were injected into mice bearing MCF7 tumor xenografts, and tumor cells labeled with one or both markers were recognized in tumor sections (in relation to DioC7+ve functional tumor blood vessels) using appropriate fluorescence-labeled antibodies and imunohistochemistry. Proliferating cells were identified by an antibody to Ki67. Mice were treated with pimo and then either doxorubicin or saline one hour later; EF5 was given after a variable interval of 24, 48, 72, 96 or 120 hours; mice were killed two hours after the second injection. Changes in the location, proliferation and oxygen status of formerly hypoxic (pimo+ve) cells were quantified by their distance from blood vessels, Ki67 status and uptake of EF5 as a function of time. Results: Following treatment with doxorubicin, the proportion of hypoxic cells in the entire tumor decreased from1.5% (pimo+ve) prior to injection to 0.7% (EF5+ve) at 24 hours. The proportion of pimo+ve formerly hypoxic cells that are no longer hypoxic (i.e. EF5 - ve) at 24 hours was 75% after treatment compared to 18% in controls indicating rescue of previous hypoxic cells that would have died in the absence of treatment. The proportion of these pimo+ve cells that were cycling (Ki67+ve) increased from 4.7% to 15.0% at 24 Hours and then slowly decreased. Conclusions: There is a decrease in the proportion of hypoxic cells in MCF7 xenografts following treatment with doxorubicin. Originally hypoxic cells (that may have died in the absence of treatment) can move closer to blood vessels, re-oxygenate, and repopulate the tumor. Treatment directed to killing hypoxic cells (e.g. with hypoxia activated pro-drugs) has the potential to improve the outcome of chemotherapy by inhibiting tumor cell repopulation. Supported by are search grant from the Canadian Institutes for Health Research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5247. doi:1538-7445.AM2012-5247

Densely Granulated Murine NK Cells Eradicate Large Solid Tumors

Natural killer (NK) cells inhibit early stages of tumor formation, recurrence, and metastasis. Here, we show that NK cells can also eradicate large solid tumors. Eradication depended on the massive infiltration of proliferating NK cells due to interleukin 15 (IL-15) released and presented by the cancer cells in the tumor microenvironment. Infiltrating NK cells had the striking morphologic feature of being densely loaded with periodic acid-Schiff-positive, diastase-resistant granules, resembling uterine NK cells. Perforin-mediated killing by these densely granulated NK cells was essential for tumor eradication. Expression of the IL-15 receptor α on cancer cells was needed to efficiently induce granulated NK cells, and expression on host stromal cells was essential to prevent tumor relapse after near complete destruction. These results indicate that IL-15 released at the cancer site induces highly activated NK cells that lead to eradication of large solid tumors.

Muramyl Dipeptide modulates differentiation, maturity of dendritic cells and anti-tumor effect of DC-mediated T cell in acute leukemia children

Background:Targeted therapy is a potentially useful approach for antileukemic therapy, in particular eliminating minimal residual disease(MRD) to prevent tumor relapse. This study was aimed to find out an effective, nontoxic dendritic cell (DC) maturating agent for the immunotherapy of acute leukemia. Results: MDP-matured DCs(M-DCs) expressed higher level of phenotypic markers and secreted higher cytokine level, while lower than TNF-α-matured DCs (T-DCs) and co-administration of MDP and TNF-α-matured DCs (MT-DCs). MT-DCs promoted significantly allogeneic T-cells reaction. As a result, allogeneic T-cell proliferated significantly and secreted higher amount of IFN-γ. HL60-derived antigens were presented more effectively by MT-DCs to cytotoxic T lymphocytes (CTLs) to induce more beneficial anti-tumor effects in a dose-dependent manner. Methods: Purified mononuclear cells (MNCs) from bone marrow of acute leukemia children were differentiated by granulocyte-macrophage colony stimulating factor (GM-CSF) and recombinant human interleukin-4 (rhIL-4) and further matured by either Muramyl Dipeptide（MDP), tumor necrosis factor-alpha (TNF-α) or co-administration of MDP and TNF-α. Conclusions: These results demonstrate MDP can be used as a candidate clinical agent for antigen specific cancer immunotherapy.

Preparation and identification of a novel immunomodulator composed of muramyl dipeptide and anti-CD10 monoclonal antibody for treatment of minimal residual disease in acute leukemia children

Targeted therapy is a potentially useful approach for antileukemic therapy, in particular eliminating minimal residual disease (MRD) to prevent tumor relapse. In this study, the immunomodulator (MDP-Ab) was constructed by coupling anti-CD10 monoclonal antibody (MAb) and muramyl dipeptide (MDP) using heterobifunctional reagent SPDP and the activity of MDP-Ab through dendritic cells (DCs)-based immunotherapy was identified in targeted therapy for leukemia. Results showed that the molecular ratio of purified MDP-Ab immunomodulator was about 2:1 according to electrospray ionization mass spectrometry (ESI-MS). The immunoreactivity and specificity of the new immunomodulator on CD10 antigen was identical to that of unconjugated native anti-CD10 MAb. The immunomodulatory effect of MDP-Ab immunoconjugate on peripheral blood dendritic cells (PBDCs) from children with acute lymphoblastic leukemia (ALL) exhibited upregulated expression of HLA-DR, co-stimulatory marker (CD80 and CD86) and maturity marker (CD83), increased cytokine secretion (interleukin-12, IL-12) and enhanced autostimulatory activity. These results in vitro suggested that MDP-Ab immunoconjugate may be a suitable candidate for targeting trials and support the further development of vaccination with the new immunomodulator-triggered DCs as a post-remission treatment to prevent relapse in ALL children.

The effect of selenium on the oncogenic protein β-catenin in human colorectal cancers.

e13553 Background: β-catenin is a multifunctional oncogenic protein leads to many types of human cancers by affecting cell growth and adhesion. Recently, Wnt/β-catenin signaling pathway is implicated in the self-renewal of stem cells. Cancer stem cells (CSCs) contribute to tumor bulk, recurrence and resistance to chemotherapy. In colorectal carcinomas, Wnt/β-catenin signaling pathway is activated due to a mutation in the tumor suppresser gene, APC. This study is to determine whether pharmacological doses of a selenium-containing compound, methylseleninic acid (MSeA), have an effect on the expression of β-catenin in colorectal carcinomas. Methods: Two human colorectal cancer cell lines, HCT-8 and HT-29,were treated with MSeA alone, SN-38 alone (the active metabolite of irinotecan, a topoisomearse I inhibitor) and in sequential combination of MSeA/SN-38. MSeA was applied at various doses and exposure times. SN-38 was used at concentration that inhibits 50% of cell growth. After treatments, the effect on intracellular level of β-catenin was detected using western blot analyses. The effect on cell growth/death was measured using Sulforhodamine B (SRB) assay. Results: Untreated HCT-8 and HT-29 cells expressed β-catenin. MSeA inhibited intracellular level of β-catenin in dose and time dependent manners. The reduced level of β-catenin after MSeA treatments was associated with inhibition of tumor cell growth and enhancement of cell death. SN-38 alone had no effect onβ-catenin level. The sequential combination of MSeA/SN-38 resulted in more inhibition of β-catenin when compared to all other treatment groups. Conclusions: In colorectal carcinomas, MSeA is a potent inhibitor of β-catenin that consequently led to cell growth inhibition and death. Sequential combination of MSeA/SN-38 resulted in further increase in β-catenin inhibition, thus offering the potential for reversal of drug resistance and tumor relapse. Based on these results and given the important role of Wnt/β-catenin signaling pathway in CSCs, MSeA is an excellent candidate regimen for future therapeutic combinations to reduce tumor bulk, reverse drug resistance and eradicate CSCs, thus preventing tumor relapse and increasing cure rates. No significant financial relationships to disclose.

Abstract 4282: The role of glycosphingolipids in the formation of cancer stem cells and drug resistance

Abstract Tumorigenesis and malignant progression of cancers rely on a small subset of cells known as cancer stem cells (CSCs). In addition to forming primary tumors, metastasis and relapse, CSCs always display resistance to cytotoxins and this resistant feature protects CSCs during the course of chemotherapy. Glucosylceramide synthase (GCS) is a limited enzyme catalyzing the first glycosylation in glycosphingolipids synthesis. Glycosphingolipids are likely to play an essential role in maintaining the stemness of stem cells, since that deletion of GCS induces apoptosis of embryonic stem cell and stops embryo development in GCS knockout mice. The stage-specific embryonic antigens (SSEA-3, SSEA-4) are glycosphingolipids and serve as common pluripotent markers for human embryonic stem cells. In the present study, we have found that GCS modulates the formation and maintenance of breast cancer stem cells. GCS overexpression was interrelated to the increase of breast cancer stem cells (BCSCs) and drug resistance in human breast cancer MCF-7 cell lines after doxorubicin induction. In MCF-7/Dox (doxorubicin-resistant) cells, GCS protein was increased by 39-fold accompanied with enhanced enzyme activity, as compared to wild-type MCF-7 cell. Analyzed by using flow cytometry and immunostaining, the BCSCs with CD44+/CD24-/ESA+ phenotype were increased by 5-fold, and 3-fold in MCF-7/Dox and NCI-ADR/RES cell lines, as compared to MCF-7 cell lines, respectively. In BCSCs, GCS enzyme activity was 2-fold greater than other populations sorted from MCF-7/Dox. Silencing GCS by using mixed-backbone oligonucleotide (MBO-asGCS) significantly decreased the numbers of BCSCs in MCF-7/Dox cells. We observed tumorigenesis of NCI/ADR-RE/GCS (GCS overexpressed) and NCI/ADR-RE/asGCS (GCS silenced) in athymic nude mice. Aggressive tumors were found in all mice inoculated with NCI/ADR-RES and NCI/ADR-RE/GCS cells that were BCSC-enriched; however, no tumor or metastasis was observed in mice injected with NCI-ADR/asGCS cells. Furthermore, MBO-asGCS treatment (1 mg/kg) significantly decreased the numbers of BCSCs isolated from tumors of NCI-ADR/RES, as compared with control groups. Furthermore, BCSCs after MBO-asGCS treatment were more sensitive to doxorubicin. These results, for the first time, demonstrate that glycosphingolipid is involved in the regulation of cancer stem cell formation. Silencing GCS eliminates BCSCs, that can reverses drug resistance as well as prevent tumor relapse. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4282.

Hydroxyurea induces a senescent, non-malignant, immunogenic state in neuroblastoma cells – a new therapeutic strategy?

Oncogene inactivation can cause cellular senescence, a permanent state of proliferative arrest, resulting in tumor “regression“. In MYCN-amplified (MNA) neuroblastoma (NB) long-term hydroxyurea (HU) treatment leads to the elimination of episomal MNA and senescence induction in vitro. The aim was to characterize senescent NB cells and describe their functional role. MLPA and arrayCGH confirmed that MNA is the only chromosomal aberration that changes between non-senescent and senescent NB-cell lines. Gene expression profiling revealed that senescent NB cells down-regulate unfavorable (e.g. survivin) and up-regulate favorable tumor markers (e.g. NTRK1, CD44). Furthermore, inhibitors of the cell cycle (p21, p15, p16), tumor growth- and angiogenesis (e.g. TIMP1, IGFBPs) were increased. In functional in vitro analyses senescent tumor cells reduced cell growth of non-senescent tumor cells and – in line with increased expression of MHCI and other immune-response-related molecules – senescent NB cells allowed T-cell and NKT-cell activation. Thus, HU induces a senescent, non-malignant, immunogenic state providing the basis for future studies using HU in NB patients to prevent tumor relapse.

Therapeutic effects of ablative radiation on local tumor require CD8+ T cells: changing strategies for cancer treatment

AbstractPatients with locally advanced cancer or distant metastasis frequently receive prolonged treatment with chemotherapy and/or fractionated radiotherapy (RT). Despite the initial clinical response, treatment resistance frequently develops and cure in these patients is uncommon. Developments in RT technology allow for the use of high-dose (or ablative) RT to target local tumors, with limited damage to the surrounding normal tissue. We report that reduction of tumor burden after ablative RT depends largely on T-cell responses. Ablative RT dramatically increases T-cell priming in draining lymphoid tissues, leading to reduction/eradication of the primary tumor or distant metastasis in a CD8+ T cell–dependent fashion. We further demonstrate that ablative RT-initiated immune responses and tumor reduction are abrogated by conventional fractionated RT or adjuvant chemotherapy but greatly amplified by local immunotherapy. Our study challenges the rationale for current RT/chemotherapy strategies and highlights the importance of immune activation in preventing tumor relapse. Our findings emphasize the need for new strategies that not only reduce tumor burden but also enhance the role of antitumor immunity.