Necrotic Tumor Cells Research Articles

Normal Lymphocytes at Diagnosis and during Induction Chemotherapy in Childhood Acute Lymphoblastic Leukaemia in Peripheral Blood and Bone Marrow Are Predominantly CD3+, CD4+ or CD8+, CCR7+, CD45RA+, HLA-DR-

Tumour immune surveillance requires both the recognition of malignant cells as immunogenic and the functional cytotoxic capacity of an effector cell. Key cytotoxic effector cells are T lymphocytes, capable of mediating tumour cell death through granule exocytosis and death receptor signalling. Whether chemotherapy can modify immune surveillance in haematological malignancies is not yet well understood. Remission induction chemotherapy induces rapid cytoreduction of leukaemia and has the modifying potential to affect effector-target ratios and tumour cell recognition. Professional antigen presenting cells may take up the necrotic and apoptotic tumour cells, process tumour antigens, and present these to T lymphocytes alongside the costimulatory molecules required for effective priming. Thus, the rapid leukaemia cytoreduction of induction therapy may allow the immune system to exert sufficient control as to sustain remission, or to achieve tumour elimination. In this study, we aimed to characterise circulating T lymphocytes and those from the bone marrow (BM) tumour microenvironment, of paediatric B cell acute lymphoblastic leukaemia (ALL) patients at diagnosis and throughout remission induction chemotherapy.17 paediatric patients diagnosed with precursor B cell ALL at Alder Hey Children's Hospital between August 2014 and February 2015 were enrolled in this study. Diagnosis, risk group assignment and treatment were performed according to the UKALL2011 multicentre trials. Philadelphia chromosome positive B-ALL or ALL in patients with Down syndrome were excluded. This study was approved by Norfolk Research Ethics Committee (REC 14/EE/0211) and informed consent was obtained from patients and/or their legal guardians in accordance with the Declaration of Helsinki. 87 paediatric BM and peripheral blood (PB) tissue samples were collected from enrolled patients at time points co-ordinating with treatment protocol (diagnosis, and induction days 8 and/or 15 and 29). CD4+ and CD8+ T cells, invariant natural killer T cells (iNKTs), cytokine induced killer T cells (CIKs) and γd T cells were stained with fluorochrome conjugated monoclonal antibody panels to allow subset analysis and activation state determination by six-colour flow cytometry.The relative frequencies of all major T cell populations; CD4+, CD8+, iNKTs, CIKs and γd T cells, were similar in PB and BM aspirate at diagnosis. CD4+ cells accounted for approximately 53% of circulating PB T cells and 47% of BM T cells. CD8+ cells accounted for approximately 38% of circulating PB T cells and 42% of BM T cells, the remaining 9% of circulating T cells and 11% of BM T cells were made up of combined iNKTs, CIKs and γd T cells. Naïve CD4+ and naïve CD8+ cells dominated both the circulating PB and BM T cell pools at diagnosis and throughout induction chemotherapy (Figure 1). Naïve CD4+ cells accounted for approximately 42% of circulating T cells and approximately 36% of BM T cells at diagnosis (Figure 1A). While naïve CD8+ cells accounted for approximately 21% of circulating T cells and 20% of BM T cells at diagnosis (Figure 1B). No significant changes in the relative frequencies of naïve CD4+ and CD8+ T cells were observed through induction days 8 and 15, although by induction day 29 the relative frequency of activated naïve CD4+ cells (HLA-DR+) was increased in the BM T cell pool compared to circulating naïve CD4+ cells in PB.T cell reconstitution following induction chemotherapy may have important implications in childhood leukaemia, especially if recovering T cells are able to mediate anti-leukaemia effects. Rapid lymphocyte recovery has been associated with improved survival and is accounted for by increasing numbers of T lymphocytes following induction therapy in paediatric ALL. Whether anti-leukaemia T cell cytotoxicity develops following induction therapy remains to be elucidated although there is the potential for a cytotoxic response to develop in both CD4+ and CD8+ T cell subsets. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The clinical role of 'liquid biopsy' in hepatocellular carcinoma.

Circulating free tumor DNA (ctDNA) is DNA released from necrotic or apoptotic tumor cells into the bloodstream. Absolute levels of ctDNA, as well as genetic mutations and epigenetic changes detected in ctDNA are useful biomarkers of tumor biology, progression and response to therapy in many tumor types and recent evidence suggests they may be useful in hepatocellular carcinoma (HCC). ctDNA detected in blood, therefore, offers a minimally invasive, easily repeated 'liquid biopsy' of cancer, providing real-time dynamic analysis of tumor behavior and treatment response that could revolutionize both clinical and research practice in HCC. In this review, we provide a critical summary of the evidence for the utility of ctDNA as a diagnostic and prognostic biomarker in HCC.

Improvement of DC vaccine with ALA-PDT induced immunogenic apoptotic cells for skin squamous cell carcinoma

Dendritic cell (DC) based vaccines have emerged as a promising immunotherapy for cancers. However, most DC vaccines so far have achieved only limited success in cancer treatment. Photodynamic therapy (PDT), an established cancer treatment strategy, can cause immunogenic apoptosis to induce an effective antitumor immune response. In this study, we developed a DC-based cancer vaccine using immunogenic apoptotic tumor cells induced by 5-aminolevulinic acid (ALA) mediated PDT. The maturation of DCs induced by PDT-treated apoptotic cells was evaluated using electron microscopy, FACS, and ELISA. The anti-tumor immunity of ALA-PDT-DC vaccine was tested with a mouse model. We observed the maturations of DCs potentiated by ALA-PDT treated tumor cells, including morphology maturation (enlargement of dendrites and increase of lysosomes), phenotypic maturation (upregulation of surface expression of MHC-II, DC80, and CD86), and functional maturation (enhanced capability to secrete IFN-γ and IL-12, and to induce T cell proliferation). Most interestingly, PDT-induced apoptotic tumor cells are more capable of potentiating maturation of DCs than PDT-treated or freeze/thaw treated necrotic tumor cells. ALA-PDT-DC vaccine mediated by apoptotic cells provided protection against tumors in mice, far stronger than that of DC vaccine obtained from freeze/thaw treated tumor cells. Our results indicate that immunogenic apoptotic tumor cells can be more effective in enhancing a DC-based cancer vaccine, which could improve the clinical application of PDT-DC vaccines.

Formylpeptide receptor 1 mediates the tumorigenicity of human hepatocellular carcinoma cells

abstractG protein-coupled chemoattractant receptors (GPCRs) have been implicated in cancer progression. Formylpeptide receptor 1 (FPR1) was originally identified as a GPCR mediating anti-microbial host defense. However, the role of FPR1 in tumorigenesis remains poorly understood. The current study aims to investigate the potential of FPR1 to regulate human hepatoma growth and invasion. We found the FPR1 gene and protein expression in human intratumoral and peritumoral tissues of hepatocellular carcinoma (HCC) specimens and in human hepatoma cell lines. FPR1 activation mediated the migration, calcium mobilization and ERK-dependent IL-8 production by hepatic cancer cells. FPR1 knockdown substantially reduced the tumorigenicity of hepatoma cells in nude mice. Necrotic hepatic tumor cells released factor(s) that activated FPR1 in live tumor cells. Our results indicate a critical role of FPR1 in the progression of malignant human hepatic cancer. FPR1 thus may represent a molecular target for the development of novel anti-hepatoma therapeutics.

The role of cell lysis and matrix deposition in tumor growth modeling

The multiphase model for tumor growth, proposed by the authors in previous works, is here enhanced. The original model includes a solid phase, the extracellular matrix (ECM) and three fluid phases: living and necrotic tumor cells (TCs), host cells (HCs), and the interstitial fluid (IF). We introduce the mathematical model for deposition (remodeling) of ECM during the TCs growth, and lysis. Differently from the previous version of the model we take into account that TCs growing in vitro depose their own ECM not present at the beginning. The lysis re-transforms the necrotic cells into IF. The updated mathematical formulation is discretized by means of the finite element method and implemented in a general purpose code. First we reproduce new experimental data of multicellular tumor spheroid (MTS) growth in vitro. The free boundary conditions used in this simulation together with necrosis and lysis allow following the tumor growth curve up to the final steady-state. The second example, by comparing results of tumor growth in an ECM-free medium and in an ECM remodeling medium highlights how ECM deposition affects tumor growth. In an initially ECM-free medium the tumor is unobstructed and can proliferate more rapidly both without ECM and in case of ECM deposition. The third example shows the effect of lysis: it redirects some tumor cells toward the necrotic core of the MTS and produces outflow of the IF from the tumor mass. The introduction of lysis and ECM deposition allows capturing different aspects of the avascular tumor growth not yet comprised in the original model: the MTS growth seems to be influenced by ECM deposition and the lysis seems to be a cause of an outflow of the IF from the tumor mass.

Abstract 5241: Clinical relevance of circulating tumor DNA in plasma from pancreatic cancer patients

Abstract Background: Circulating tumor DNA (ctDNA) may originate from necrotic or apoptotic tumor cells in the primary tumor, metastatic lesions or in the circulation. There is evidence that the ctDNA level may reflect the total tumor burden in a patient. We wanted to investigate whether changes in the ctDNA level might be used to monitor disease progression in pancreatic cancer patients with locally or advanced disease. Methods: Blood samples (9 mL in EDTA tubes) were collected from 15 prospectively recruited patients with locally advanced and/or metastatic pancreatic cancer before initiation of treatment, and subsequently every month during chemotherapy. The samples were processed by LymphoprepTM (Axis Shield) density centrifugation before plasma DNA isolation using the QIAamp Circulating Nucleic Acid kit (Qiagen). A high-fidelity polymerase-based PNA clamp PCR method (Gilje et al., 2008, Oltedal et al., 2010) was then used for the detection of KRAS mutations in exon 12 and 13, as a surrogate marker for ctDNA. KRAS mutations have previously been reported to be present in >90% of the pancreatic cancers. Plasma samples from 29 healthy individuals were also analysed as a reference group for the PNA clamp PCR method. The results were compared with conventional biochemical and radiological monitoring measures. Results: The majority of the patients (80%) had metastatic disease and they were treated either by gemcitabine or FOLFORINOX. Nine (60%) patients had a positive KRAS status in the plasma samples obtained before initiation of chemotherapy, indicating presence of ctDNA. Moreover, 11 of the 15 included patients had ≥2 follow-up samples, and in several of these patients the ctDNA level changed substantially during the course of chemotherapy. In total, 17/38 (44.7%) patient samples were positive for plasma DNA KRAS mutations during chemotherapy. Changes in the ctDNA level seemed to correspond both to radiological follow-up data and changes in CA19-9 for several patients. Analyses of the total plasma DNA fraction with regard to disease monitoring, gave more inconclusive results in this small pilot study. Conclusion: Our pilot study gives support to the hypothesis that ctDNA may be used as a marker for monitoring treatment efficacy and disease progression in pancreatic cancer patients. This will be further investigated. Citation Format: Kjersti Tjensvoll, Morten Lapin, Tove Buhl, Satu Oltedal, Katrine Steen-Ottosen Berry, Bjørnar Gilje, Jon Arne Søreide, Millind Javle, Oddmund Nordgård, Rune Smaaland. Clinical relevance of circulating tumor DNA in plasma from pancreatic cancer patients. [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 5241. doi:10.1158/1538-7445.AM2015-5241

Specific cellular immune response elicited by the necrotic tumor cell-stimulated macrophages.

To determine whether the necrotic tumor cell-stimulated macrophages (NTCSM) could elicit specific immune response. Mice were immunized with the necrotic H22 tumor cell lysate-stimulated macrophages and the specific immune responses against the same tumor challenge were examined. The morphologic characteristics were observed with the transmission electron microscope and scanning electron microscopy. The expression of CD14, CD68, CD80 and CD86 were detected with the flow cytometer. The cytotoxicity and cytokine production of splenocytes were measured with the MTT assay and ELISA assay respectively. Our research results reveal that NTCSMs are larger cells which generally generate spherical and elongated protrusions, folding membrane, and vesicles on their surface. Also, abundant lysosomes, secondary lysosomes, phagosomes, rough endoplasmic reticulum, and lipid bodies were found in their cytoplasm. The flow cytometry results show that the necrotic H22 tumor cell lysate could enhance the expression of CD14 and CD86 molecules and the NTCSM was characterized by the expression of CD14+/-CD68+CD80-CD86+. After the mice were vaccinated with NTCSMs, the tumor forming rate, tumor volume and weight of the NTCSM-vaccinated group were significantly lower than those of the sterile saline-injected group and untreated macrophage-vaccinated group (p<0.05). The cytotoxicity to H22 tumor cells of the splenocytes obtained from the NTCSM-immunized group was higher than that of the sterile saline-injected group and untreated macrophage-vaccinated group (p<0.05). Meanwhile, the levels of IL-2 and IFN-γ in the culture supernatant of the NTCSM-immunized group were higher significantly than those of the saline-injected group and untreated macrophage-vaccinated group. The level of IL-4 of the NTCSM-immunized group was significantly lower than those of the other two groups. Our results indicated that NTCSMs could elicit specific cellular immune responses in vivo.

Improvement of DC vaccine with ALA-PDT induced immunogenic apoptotic cells for skin squamous cell carcinoma.

Dendritic cell (DC) based vaccines have emerged as a promising immunotherapy for cancers. However, most DC vaccines so far have achieved only limited success in cancer treatment. Photodynamic therapy (PDT), an established cancer treatment strategy, can cause immunogenic apoptosis to induce an effective antitumor immune response. In this study, we developed a DC-based cancer vaccine using immunogenic apoptotic tumor cells induced by 5-aminolevulinic acid (ALA) mediated PDT. The maturation of DCs induced by PDT-treated apoptotic cells was evaluated using electron microscopy, FACS, and ELISA. The anti-tumor immunity of ALA-PDT-DC vaccine was tested with a mouse model. We observed the maturations of DCs potentiated by ALA-PDT treated tumor cells, including morphology maturation (enlargement of dendrites and increase of lysosomes), phenotypic maturation (upregulation of surface expression of MHC-II, DC80, and CD86), and functional maturation (enhanced capability to secrete IFN-γ and IL-12, and to induce T cell proliferation). Most interestingly, PDT-induced apoptotic tumor cells are more capable of potentiating maturation of DCs than PDT-treated or freeze/thaw treated necrotic tumor cells. ALA-PDT-DC vaccine mediated by apoptotic cells provided protection against tumors in mice, far stronger than that of DC vaccine obtained from freeze/thaw treated tumor cells. Our results indicate that immunogenic apoptotic tumor cells can be more effective in enhancing a DC-based cancer vaccine, which could improve the clinical application of PDT-DC vaccines.

Hoechst 33258–conjugated hyaluronated fullerene for efficient photodynamic tumor therapy and necrotic tumor targeting

In this study, we synthesized a Hoechst 33258–conjugated hyaluronated fullerene consisting of Hoechst 33258 (as a target moiety to detect necrotic tumor cells), hyaluronic acid (as a target polymer to bind the CD44 receptor overexpressed on the surface of tumor cells), and fullerene (as a photosensitizing agent). This conjugate self-assembled to form nanoparticles consisting of a hydrophilic block (Hoechst 33258 and hyaluronic acid) and a lipophilic block (fullerene). We utilized these nanoparticles to improve the antitumor efficacy via photodynamic tumor therapy. The HCT-116 cells that were damaged after the first photodynamic tumor therapy (using hyaluronated fullerene nanoparticles) were again targeted using Hoechst 33258–conjugated hyaluronated fullerene nanoparticles (detecting necrotic tissues). The experimental results revealed that the second photodynamic tumor therapy using Hoechst 33258–conjugated hyaluronated fullerene nanoparticles caused significant increases in the in vitro phototoxicity and the in vivo tumor inhibition, thereby suggesting their pharmaceutical potential for designing effective multiple photodynamic tumor therapy treatments.

Preparation of dendritic cells for cancer immunotherapy

Development of new effective method for cancer therapy is one of the most important trends in the modern medicine. Along with surgery, chemotherapy and radiotherapy, induction of an immune response against the tumor cells is a promising approach for therapy of cancer, particularly metastatic, slowly dividing tumors and cancer stem cells. Induction of the antitumor T-cell immune response involves activation of antigen-presenting cells, which can efficiently present the cancer antigens and activate T-lymphocytes. The immune response may be activated by dendritic cells (DC) loaded with tumor antigens, such as tumor-specific proteins, tumor cell lysates, apoptotic or necrotic tumor cells, as well as nucleic acids encoding tumor antigens. Regardless of the selected source of the tumor antigen, preparation of mature DC is a principal step in the development of anticancer vaccines aimed at the induction of the cytotoxic T-cell immune response. Recently, various research groups have proposed several strategies for producing mature DC, differed by the set of agents used. It has been shown that the maturation strategy influences both their phenotype and the ability to induce the immune response. In this review we have analyzed the results of studies on the various strategies of preparation of mature DCs.

Targeted α-therapy using 227Th-APOMAB and cross-fire antitumour effects: preliminary in-vivo evaluation.

Resistance to conventional cancer treatments is a major problem associated with solid tumours. Tumour hypoxia is associated with a poor prognosis and with poor treatment outcomes; therefore, there is a need for treatments that can kill hypoxic tumour cells. One potential option is targeted α-radioimmunotherapy, as α-particles can directly kill hypoxic tumour cells. The murine monoclonal antibody DAB4 (APOMAB), which binds dead tumour cells after DNA-damaging treatment, was conjugated and radiolabelled with the α-particle-emitting radionuclide thorium-227 (Th). Mice bearing Lewis lung tumours were administered Th-DAB4 alone or after chemotherapy and the tissue biodistribution of the radioimmunoconjugate was examined, as was the effect of these treatments on tumour growth and survival. Th-DAB4 accumulated in the tumour particularly after chemotherapy, whereas the distribution in healthy tissues did not change. Th-DAB4 as a monotherapy increased survival, with more pronounced responses observed when given after chemotherapy. We have shown that targeted α-therapy of necrotic tumour cells with Th-DAB4 had significant and surprising antitumour activity as it would occur only through a cross-fire effect.

Irradiation of necrotic tumor cells used to pulse dendritic cells (DCs) potentiates DC vaccine-induced anti-tumor immunity in a mouse model of high-grade glioma

Meeting abstracts The prognosis of high-grade glioma (HGG) is poor despite advancements in neurosurgery, radio-and chemotherapy. DC-based immunotherapy has emerged as a promising and feasible treatment approach due to its high degree of selectivity and its ability to induce an antigen-specific

Photodynamic Therapy for Lung Cancer and Malignant Pleural Mesothelioma

Photodynamic therapy (PDT) is a form of non-ionizing radiation therapy that uses a drug, called a photosensitizer, combined with light to produce singlet oxygen ((1)O2) that can exert anti-cancer activity through apoptotic, necrotic, or autophagic tumor cell death. PDT is increasingly being used to treat thoracic malignancies. For early-stage non-small cell lung cancer (NSCLC), PDT is primarily employed as an endobronchial therapy to definitively treat endobronchial or roentgenographically occult tumors. Similarly, patients with multiple primary lung cancers may be definitively treated with PDT. For advanced or metastatic NSCLC and small cell lung cancer (SCLC), PDT is primarily employed to palliate symptoms from obstructing endobronchial lesions causing airway compromise or hemoptysis. PDT can be used in advanced NSCLC to attempt to increase operability or to reduce the extent of operation intervention required, and selectively to treat pleural dissemination intraoperatively following macroscopically complete surgical resection. Intraoperative PDT can be safely combined with macroscopically complete surgical resection and other treatment modalities for malignant pleural mesothelioma (MPM) to improve local control and prolong survival. This report reviews the mechanism of and rationale for using PDT to treat thoracic malignancies, details prospective and major retrospectives studies of PDT to treat NSCLC, SCLC, and MPM, and describes improvements in and future roles and directions of PDT.

P1-31-3 - Radiation-Induced Leiomyosarcoma in Prostate After Brachytherapy for Prostatic Adenocarcinoma

Radiation (RTx) has been employed as a curative therapy for prostatic adenocarcinoma. RTx-induced sarcomas are rare late adverse event, representing less than 0.2% of all irradiated patients. RTx-induced sarcomas are more aggressive tumors than prostatic adenocarcinoma. Herein, we present a case with RTx-induced prostatic leiomyosarcoma after permanent brachytherapy for prostatic adenocarcinoma.A 69-year-old male was diagnosed as localized prostatic adenocarcinoma, and treated by brachytherapy. Dysuria and gross hematuria started to appear six years after brachytherapy. Urethroscopy showed stenosis by a tumor at the prostate. Transurethral prostatectomy was performed for a diagnosis. Based on pathological findings, we diagnosed as leiomyosarcoma of prostate. He was treated with three cycles of neo-adjuvant chemotherapy (CT) consisted with doxorubicin and ifosfamide (AI), and then performed prostatocystectomy with intrapelvic lymphadenectomy. The tumor extended from the prostate and infiltrated the bladder wall and serosa with lymphatic and venous invasion. The surgical margin was negative, and no residual prostatic adenocarcinoma was observed. The proportion of necrotic tumor cells by neo-adjuvant CT was less than 50%. Subsequently, adjuvant CT was offered, but he chose a follow-up without CT. Local recurrence and lung metastasis were detected by computed tomography three months after the surgery. He was treated again with AI. However, CT was not effective and he died six months after the operation. In conclusion, effective treatment strategy for prostatic sarcoma should be developed in the near future, although the clinical feature of prostatic sarcoma remains unclear due to rare incidence.

Fractionated radiotherapy is the main stimulus for the induction of cell death and of Hsp70 release of p53 mutated glioblastoma cell lines

BackgroundGlioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Despite a multimodal therapy consisting of resection followed by fractionated radiotherapy (RT) combined with the chemotherapeutic agent (CT) temozolomide (TMZ), its recurrence is almost inevitable. Since the immune system is capable of eliminating small tumor masses, a therapy should also aim to stimulate anti-tumor immune responses by induction of immunogenic cell death forms. The histone deacetylase inhibitor valproic acid (VPA) might foster this.MethodsReflecting therapy standards, we applied in our in vitro model fractionated RT with a single dose of 2Gy and clinically relevant concentrations of CT. Not only the impact of RT and/or CT with TMZ and/or VPA on the clonogenic potential and cell cycle of the glioblastoma cell lines T98G, U251MG, and U87MG was analyzed, but also the resulting cell death forms and release of danger signals such as heat-shock protein70 (Hsp70) and high-mobility group protein B1 (HMGB1).ResultsThe clonogenic assays revealed that T98G and U251MG, having mutated tumor suppressor protein p53, are more resistant to RT and CT than U87MG with wild type (WT) p53. In all glioblastoma cells lines, fractionated RT induced a G2 cell cycle arrest, but only in the case of U87MG, TMZ and/or VPA alone resulted in this cell cycle block. Further, fractionated RT significantly increased the number of apoptotic and necrotic tumor cells in all three cell lines. However, only in U87MG, the treatment with TMZ and/or VPA alone, or in combination with fractionated RT, induced significantly more cell death compared to untreated or irradiated controls. While necrotic glioblastoma cells were present after VPA, TMZ especially led to significantly increased amounts of U87MG cells in the radiosensitive G2 cell cycle phase. While CT did not impact on the release of Hsp70, fractionated RT resulted in significantly increased extracellular concentrations of Hsp70 in p53 mutated and WT glioblastoma cells.ConclusionsOur results indicate that fractionated RT is the main stimulus for induction of glioblastoma cell death forms with immunogenic potential. The generated tumor cell microenvironment might be beneficial to include immune therapies for GBM in the future.

Purified Dendritic Cell-Tumor Fusion Hybrids Supplemented with Non-Adherent Dendritic Cells Fraction Are Superior Activators of Antitumor Immunity

BackgroundStrong evidence supports the DC-tumor fusion hybrid vaccination strategy, but the best fusion product components to use remains controversial. Fusion products contain DC-tumor fusion hybrids, unfused DCs and unfused tumor cells. Various fractions have been used in previous studies, including purified hybrids, the adherent cell fraction or the whole fusion mixture. The extent to which the hybrids themselves or other components are responsible for antitumor immunity or which components should be used to maximize the antitumor immunity remains unknown.MethodsPatient-derived breast tumor cells and DCs were electro-fused and purified. The antitumor immune responses induced by the purified hybrids and the other components were compared.ResultsExcept for DC-tumor hybrids, the non-adherent cell fraction containing mainly unfused DCs also contributed a lot in antitumor immunity. Purified hybrids supplemented with the non-adherent cell population elicited the most powerful antitumor immune response. After irradiation and electro-fusion, tumor cells underwent necrosis, and the unfused DCs phagocytosed the necrotic tumor cells or tumor debris, which resulted in significant DC maturation. This may be the immunogenicity mechanism of the non-adherent unfused DCs fraction.ConclusionsThe non-adherent cell fraction (containing mainly unfused DCs) from total DC/tumor fusion products had enhanced immunogenicity that resulted from apoptotic/necrotic tumor cell phagocytosis and increased DC maturation. Purified fusion hybrids supplemented with the non-adherent cell population enhanced the antitumor immune responses, avoiding unnecessary use of the tumor cell fraction, which has many drawbacks. Purified hybrids supplemented with the non-adherent cell fraction may represent a better approach to the DC-tumor fusion hybrid vaccination strategy.

Peptidases released by necrotic cells control CD8+ T cell cross-priming

Cross-priming of CD8+ T cells and generation of effector immune responses is pivotal for tumor immunity as well as for successful anticancer vaccination and therapy. Dead and dying cells produce signals that can influence Ag processing and presentation; however, there is conflicting evidence regarding the immunogenicity of necrotic cell death. We used a mouse model of sterile necrosis, in which mice were injected with sterile primary necrotic cells, to investigate a role of these cells in priming of CD8+ T cells. We discovered a molecular mechanism operating in Ag donor cells that regulates cross-priming of CD8+ T cells during primary sterile necrosis and thereby controls adaptive immune responses. We found that the cellular peptidases dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1), both of which are present in nonimmunogenic necrotic cells, eliminated proteasomal degradation products and blocked Ag cross-presentation. While sterile necrotic tumor cells failed to induce CD8+ T cell responses, their nonimmunogenicity could be reversed in vitro and in vivo by inactivation of DPP-3 and TOP-1. These results indicate that control of cross-priming and thereby immunogenicity of primary sterile necrosis relies on proteasome-dependent oligopeptide generation and functional status of peptidases in Ag donor cells.

Antitumor immune responses induced by ionizing irradiation and further immune stimulation.

The therapy of cancer emerged as multimodal treatment strategy. The major mode of action of locally applied radiotherapy (RT) is the induction of DNA damage that triggers a network of events that finally leads to tumor cell cycle arrest and cell death. Along with this, RT modifies the phenotype of the tumor cells and their microenvironment. Either may contribute to the induction of specific and systemic antitumor immune responses. The latter are boosted when additional immune therapy (IT) is applied at distinct time points during RT. We will focus on therapy-induced necrotic tumor cell death that is immunogenic due to the release of damage-associated molecular patterns. Immune-mediated distant bystander (abscopal) effects of RT when combined with dendritic cell-based IT and the role of fractionation of radiation in the induction of immunogenic tumor cell death will be discussed. Autologous whole-tumor-cell-based vaccines generated by high hydrostatic pressure technology will be introduced and the influence of cytokines and the immune modulator AnnexinA5 on the ex vivo generated or in situ therapy-induced vaccine efficacy will be outlined. RT should be regarded as immune adjuvant for metastatic disease and as a tool for the generation of an in situ vaccine when applied at distinct fractionation doses or especially in combination with IT to generate immune memory against the tumor. To identify the most beneficial combination and chronology of RT with IT is presumably one of the biggest challenges of innovative tumor research and therapies.

Optical Imaging of Treatment-Related Tumor Cell Death Using a Heat Shock Protein-90 Alkylator

The ability to assess in near-real time the tumor cell killing efficacy of chemotherapy regimens would improve patient treatment and survival. An ineffective regimen could be abandoned early in favor of a more effective treatment. We sought to noninvasively image treatment-related tumor cell death in mice using an optically labeled synthetic heat shock protein-90 (Hsp90) alkylator, 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid (GSAO). The Hsp90 chaperone is an important element in oncogene addiction and tumor cell survival, and its expression is enhanced by chemotherapy. These factors were predicted to favor the detection of tumor cell death using GSAO. GSAO specifically labeled apoptotic and necrotic tumor cells in culture and cells of comparable morphology in subcutaneous human pancreatic carcinoma tumors in mice. A near-infrared fluorescent conjugate of GSAO was used to noninvasively image cyclophosphamide-induced tumor cell death in murine orthotopic human mammary tumors. The GSAO conjugate did not accumulate in healthy organs or tissues in the mouse, and unbound compound was excreted rapidly via the kidneys. There was a significant increase in the GSAO fluorescence signal in the treated tumors measured either in vivo or ex vivo, and the fluorescence signal colocalized with apoptotic cells in sectioned tumors. The favorable biodistribution of optically labeled GSAO, the nature of its tumor cell target, and its capacity to noninvasively detect tumor cell death should facilitate the application of this compound in studies of the efficacy of existing and new chemotherapeutics.

Is there any prognostic importance of pretreatment serum M30 and serum M65 levels on progression-free survival of patients with metastatic renal cell carcinoma treated with first-line sunitinib?

e15569 Background: Effective cancer biomarkers for early detection, prognosis, or prediction of therapy response are urgently need in metastatic renal cell cancer (RCC). Soluble cytokeratin 18 fragments (M30, M65) are released from human cancer cells during epitelial cell death. Specific enzyme-linked immunosorbent assays (ELISA) using related antibodies distinguish between apoptotic (M30) or apoptotic and necrotic (M65) tumor cell death in serum samples. The aim of this study was to determine the prognostic value of plasma M30 and M65 levels in predicting survival rates of patients with metastatic RCC treated with first-line sunitinib. Methods: Thirty-nine patients with metastatic RCC and 39 healthy controls were included in this study. The patients’ samples were collected prior to the first cycle of sunitinib therapy and serum M30 and M65 levels were measured by ELISA. Results: The median ages of the patients and controls were 60 and 58 years, respectively. No difference was detected in the median serum M30 level between the patients and controls (53.7 vs. 49.1 U/l, P = 0.31). The median serum M65 level was significantly higher in patients than in controls (334.0 vs. 179.1 U/l, P&lt;0.001). Receiver operating characteristic (ROC) analysis revealed that the best cut-off value for serum M65 level for predicting progression-free survival (PFS) was 313.6 U/l. The median PFS of patients whose M65 levels were lower than or equal to 313.6 U/l was better than that of patients whose M65 levels were greater than 313.6 U/l (P = 0.03) in univariate analysis. But serum M65 levels in patient group were not found to be an important prognostic factor for PFS in the multivariate analysis. Conclusions: Serum M65 levels were significantly elevated in patients with metastatic RCC compared to healthy individuals. Future prospective studies with large sample sizes are needed to address the possible impact of M30 and M65 levels on the treatment responses of patients and whether these markers may be prognostic factors for PFS or OS in patients with RCC.