Eradication of established tumors in mice by a combination antibody-based therapy

Tumor-cell apoptosis is the basis of many cancer therapies, and tumor-specific T cells are the principal effectors of successful antitumor immunotherapies. Here we show that induction of tumor-cell apoptosis by an agonistic monoclonal antibody to DR5, the apoptosis-inducing receptor for TNF-related apoptosis-inducing ligand (TRAIL), combined with T-cell activation by agonistic monoclonal antibodies to the costimulatory molecules CD40 and CD137, potently and rapidly stimulated tumor-specific effector CD8+ T cells capable of eradicating preestablished tumors. Primary fibrosarcomas initiated with the carcinogen 3-methylcholanthrene (MCA), multiorgan metastases and a primary tumor containing as many as 90% tumor cells resistant to DR5-specific monoclonal antibody were rejected without apparent toxicity or induction of autoimmunity. This combination therapy of three monoclonal antibodies (trimAb) rapidly induced tumor-specific CD8+ T cells producing interferon (IFN)-gamma in the tumor-draining lymph node, consistent with a crucial requirement for CD8+ T cells and IFN-gamma in the tumor rejection process. These results in mice indicate that a rational monoclonal antibody-based therapy that both causes tumor-cell apoptosis through DR5 and activates T cells may be an effective strategy for cancer immunotherapy in humans.     

Defective immunogenic cell death of HMGB1-deficient tumors: compensatory therapy with TLR4 agonists

Immunogenic cell death induced by anticancer chemotherapy is characterized by a series of molecular hallmarks that include the exodus of high-mobility group box 1 protein (HMGB1) from dying cells. HMGB1 is a nuclear nonhistone chromatin-binding protein. It is secreted at the late stages of cellular demise and engages Toll-like receptor4 (TLR4) on dendritic cells (DCs) to accelerate the processing of phagocytic cargo in the DC and to facilitate antigen presentation by DC to T cells. The absence of HMGB1 expression by dying tumor cells exposed to anthracyclines or oxaliplatin compromises DC-dependent T-cell priming by tumor-associated antigens. Here, we show that transplantable tumors exhibiting weak expression of nuclear HMGB1 respond to chemotherapy more effectively if the treatment is combined with the local or systemic administration of a highly purified and physiochemically defined and standardized lipopolysaccharide solution, which acts as a high-potency and exclusive TLR4 agonist, called Dendrophilin (DEN). The synergistic antitumor effects mediated by the combination of chemotherapy and immunotherapy relied upon the presence of the MyD88 (myeloid differentiation primary response gene) adapter of TLR4 (but not that of the TIR-domain-containing adapter-inducing interferon-β adapter), in line with the well-characterized action of DEN on the MyD88 signaling pathway. DEN and anthracyclines synergized to induce intratumoral accumulation of interferon-γ-producing CD4⁺ and CD8⁺ T lymphocytes. Moreover, DEN could restore the immunogenicity of dying tumor cells from which HMGB1 had been depleted by RNA interference. These findings underscore the potential clinical utility of combination regimens involving immunogenic chemotherapy and certain TLR4 agonists in advanced HMGB1-deficient cancers.     

Potent antitumor effects of combination therapy with IFNs and monocytes in mouse models of established human ovarian and melanoma tumors

Interferon-activated monocytes are known to exert cytocidal activity against tumor cells in vitro. Here, we have examined whether a combination of IFN-α2a and IFN-γ and human monocytes mediate significant antitumor effects against human ovarian and melanoma tumor xenografts in mouse models. OVCAR-3 tumors were treated i.t. with monocytes alone, IFN-α2a and IFN-γ alone or combination of all three on day 0, 15 or 30 post-tumor implantation. Mice receiving combination therapy beginning day 15 showed significantly reduced tumor growth and prolonged survival including complete regression in 40% mice. Tumor volumes measured on day 80 in mice receiving combination therapy (206 mm(3)) were significantly smaller than those of mice receiving the IFNs alone (1,041 mm(3)), monocytes alone (1,111 mm(3)) or untreated controls (1,728 mm(3)). Similarly, combination therapy with monocytes and IFNs of much larger tumor also inhibited OVCAR-3 tumor growth. Immunohistochemistry studies showed a large number of activated macrophages (CD31(+)/CD68(+)) infiltrating into OVCAR-3 tumors and higher densities of IL-12, IP10 and NOS2, markers of M1 (classical) macrophages in tumors treated with combination therapy compared to the controls. Interestingly, IFNs-activated macrophages induced apoptosis of OVCAR-3 tumor cells as monocytes alone or IFNs alone did not mediate significant apoptosis. Similar antitumor activity was observed in the LOX melanoma mouse model, but not as profound as seen with the OVCAR-3 tumors. Administration of either mixture of monocytes and IFN-α2a or monocytes and IFN-γ did not inhibit Lox melanoma growth; however, a significant inhibition was observed when tumors were treated with a mixture of monocytes, IFN-α2a and IFN-γ. These results indicate that monocytes and both IFN-α2a and IFN-γ may be required to mediate profound antitumor effect against human ovarian and melanoma tumors in mouse models.     

Overcoming trastuzumab resistance in HER2-positive breast cancer using combination therapy

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC) comprises around 20–30% of all BC subtypes and is correlated with poor prognosis. For many years, trastuzumab, a monoclonal antibody, has been used to inhibit the HER2 activity. Though, the main resistance to trastuzumab has challenged the use of this drug in the management of HER2-positive BC. Therefore, the determination of resistance mechanisms and the incorporation of new agents may lead to the development of a better blockade of the HER family receptor signaling. During the last few years, some therapeutic drugs have been developed for treating patients with trastuzumab-resistant HER2-positive BC that have more effective influences in the management of this condition. In this regard, the present study aimed at reviewing the mechanisms of trastuzumab resistance and the innovative therapies that have been investigated in trastuzumab-resistant HER2-positive BC subjects.     

Curative effects of combination therapy with lentinan and interleukin-2 against established murine tumors,and the role of CD8-positive T cells

The antitumor activity of a combination of an antitumor polysaccharide, lentinan (a 1–3 glucan with 1–6 branches), and interleukin-2 (IL-2) was evaluated against established MBL-2 lymphoma and S908.D2 sarcoma at i.d. sites. Treatment of the MBL-2-tumor-bearing BDF1 mice with lentinan and IL-2 induced complete regression of tumor in 87.5% of mice treated. In contrast, treatments using either lentinan or IL-2 alone failed to induce complete regression of tumor, although temporal growth inhibition of tumor was observed about in half of the mice treated. Improvements of antitumor effects by the combination of lentinan and IL-2 were also observed in the MBL-2/B6 and S908.D2/B10.D2 systems. Expression of the antitumor effects of lentinan/IL-2 treatments required the intact T cell compartment, because the effects were not observed when nude mice were used. In the MBL-2/B6 system, the antitumor action of lentinan/IL-2 treatment was abolished in mice treated with antibody to CD8 antigen, whereas antibodies to CD4 or NK1.1 were ineffective. Furthermore, augmented tumor-specific cytotoxic T lymphocyte (CTL) activity was observed in regional lymph node cells of the mice after lentinan and IL-2 administration. These data indicate that the antitumor effects of lentinan/IL-2 are mediated by CD8⁺ CTL but not by CD4⁺ T cells or NK1.1⁺ NK/LAK cells, and suggest that this combined therapy may be effective against even established tumors that are resistant to IL-2 therapy.Abbreviations B6 C57BL/6 - BDF1 C57BL/6 × DBA/2 F1 - Lyt2 murine CD8, Lyt2.1. allele of murine CD8 - Lyt2.2 allele of murine CD8 - Lyt3 murine CD8 - L3T4 murine CD4     

Expression of the apoptosis-inducing ligands FasL and TRAIL in malignant and benign human breast tumors

Apoptosis-inducing ligands such as Fas ligand (FasL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) have been found to play an important role in cell regulation. Different malignant tumors show an altered expression of these ligands and their respective receptors compared to normal tissues. The purpose of this study was therefore to investigate expression of TRAIL, FasL, and its receptor Fas on protein and mRNA levels in breast carcinomas (n=40), fibroadenomas (n=7), and normal breast tissues (n=5). Immunohistochemical reaction demonstrated that FasL was strongly expressed in breast cancer tissues (34/40) while only one fibroadenoma and one normal breast tissue reacted weakly positive for FasL. All fibroadenomas and normal breast tissues as well as the majority of breast cancer tissues expressed Fas on protein level. Quantitative RT-PCR analysis detected high expression of FasL mRNA in breast cancer tissues and fibroadenomas, whereas fibroadenomas showed the highest Fas mRNA copy numbers, followed by breast cancer tissues and normal breast tissues (P<0.05). Compared to FasL expression, TRAIL could be detected in less breast cancer tissues on protein level (21/40) and was found in only one fibroadenoma and none of the normal breast tissues. Thus, it can be concluded that malignant breast tumors show an altered expression of the two apoptosis-inducing ligands FasL and TRAIL. Accepted: 4 January 2000     

SAHA/TRAIL combination induces detachment and anoikis of MDA-MB231 and MCF-7 breast cancer cells

SAHA, an inhibitor of histone deacetylase activity, has been shown to sensitize tumor cells to apoptosis induced by TRAIL, a member of TNF-family. In this paper we investigated the effect of SAHA/TRAIL combination in two breast cancer cell lines, the ERα-positive MCF-7 and the ERα-negative MDA-MB231. Treatment of MDA-MB231 and MCF-7 cells with SAHA in combination with TRAIL caused detachment of cells followed by anoikis, a form of apoptosis which occurs after cell detachment, while treatment with SAHA or TRAIL alone did not produce these effects. The effects were more evident in MDA-MB231 cells, which were chosen for ascertaining the mechanism of SAHA/TRAIL action. Our results show that SAHA decreased the level of c-FLIP, thus favouring the interaction of TRAIL with the specific death receptors DR4 and DR5 and the consequent activation of caspase-8. These effects increased when the cells were treated with SAHA/TRAIL combination. Because z-IEDT-fmk, an inhibitor of caspase-8, prevented both the cleavage of the focal adhesion-kinase FAK and cell detachment, we suggest that activation of caspase-8 can be responsible for both the decrement of FAK and the consequent cell detachment. In addition, treatment with SAHA/TRAIL combination caused dissipation of ΔΨ(m), activation of caspase-3 and decrement of both phospho-EGFR and phospho-ERK1/2, a kinase which is involved in the phosphorylation of BimEL. Therefore, co-treatment also induced decrement of phospho-BimEL and a concomitant increase in the dephosphorylated form of BimEL, which plays an important role in the induction of anoikis. Our findings suggest the potential application of SAHA in combination with TRAIL in clinical trials for breast cancer.     

Toll-like receptor (TLR)2 and TLR4 agonists regulate CCR expression in human monocytic cells

Interactions between proinflammatory and cell maturation signals, and the pathways that regulate leukocyte migration, are of fundamental importance in controlling trafficking and recruitment of leukocytes during the processes of innate and adaptive immunity. We have investigated the molecular mechanisms by which selective Toll-like receptor (TLR)2 and TLR4 agonists regulate expression of CCR1 and CCR2 on primary human monocytes and THP-1 cells, a human monocytic cell line. We found that activation of either TLR2 (by Pam(3)CysSerLys(4)) or TLR4 (by purified LPS) resulted in down-modulation of both CCR1 and CCR2. Further investigation of TLR-induced down-modulation of CCR1 revealed differences in the signaling pathways activated, and chemokines generated, via the two TLR agonists. TLR2 activation caused slower induction of the NF-kappa B and mitogen-activated protein kinase signaling pathways and yet a much enhanced and prolonged macrophage-inflammatory protein 1 alpha (CC chemokine ligand 3) protein production, when compared with TLR4 stimulation. Enhanced macrophage-inflammatory protein 1 alpha production may contribute to the prolonged down-regulation of CCR1 cell surface expression observed in response to the TLR2 agonist, as preventing chemokine generation with the protein synthesis inhibitor cycloheximide, or CCR1 signaling with the receptor antagonist UCB35625, abolished TLR2- and TLR4-induced CCR1 down-modulation. This result suggests an autocrine pathway, whereby TLR activation can induce chemokine production, which then leads to homologous down-regulation of the cognate receptors. This work provides further insights into the mechanisms that regulate leukocyte recruitment and trafficking during TLR-induced inflammatory responses.     

Estrogen receptor agonists/antagonists in breast cancer therapy: A critical review

Estrogens display intriguing tissue selective action that is of great biomedical importance in the development of optimal therapeutics for the prevention and treatment of breast cancer. There are also strong evidences to show that both endogenous and exogenous estrogens are involved in the pathogenesis of breast cancer. Tamoxifen has been the only drug of choice for more than 30 years to treat patients with estrogen related (ER) positive breast tumors. There is a need therefore, for identifying newer, potential and novel candidates for breast cancer. Keeping this in view, the present review focuses on selective estrogen receptor modulators and estrogen antagonists such as sulfatase and aromatase inhibitors involved in breast cancer therapy. A succinct and critical overview of the structure of estrogen receptors, their signaling and involvement in breast carcinogenesis are herein described.     

MyD88-dependent and MyD88-independent pathways in synergy, priming, and tolerance between TLR agonists

TLRs sense components of microorganisms and are critical host mediators of inflammation during infection. Different TLR agonists can profoundly alter inflammatory effects of one another, and studies suggest that the sequence of exposure to TLR agonists may importantly impact on responses during infection. We tested the hypothesis that synergy, priming, and tolerance between TLR agonists follow a pattern that can be predicted based on differential engagement of the MyD88-dependent (D) and the MyD88-independent (I) intracellular signaling pathways. Inflammatory effects of combinations of D and I pathway agonists were quantified in vivo and in vitro. Experiments used several D-specific agonists, an I-specific agonist (poly(I:C)), and LPS, which acts through both the D and I pathways. D-specific agonists included: peptidoglycan-associated lipoprotein, Pam3Cys, flagellin, and CpG DNA, which act through TLR2 (peptidoglycan-associated lipoprotein and Pam3Cys), TLR5, and TLR9, respectively. D and I agonists were markedly synergistic in inducing cytokine production in vivo in mice. All of the D-specific agonists were synergistic with poly(I:C) in vitro in inducing TNF and IL-6 production by mouse bone marrow-derived macrophages. Pretreatment of bone marrow-derived macrophages with poly(I:C) led to a primed response to subsequent D-specific agonists and vice versa, as indicated by increased cytokine production, and increased NF-kappaB translocation. Pretreatment with a D-specific agonist augmented LPS-induced IFN-beta production. All D-specific agonists induced tolerance to one another. Thus, under the conditions studied here, simultaneous and sequential activation of both the D and I pathways causes synergy and priming, respectively, and tolerance is induced by agonists that act through the same pathway.     

TLR9 agonists induced cell death in Burkitt's lymphoma cells is variable and influenced by TLR9 polymorphism

Toll-like receptor 9 (TLR9) triggering is a promising novel strategy to combat cancer as it induces innate and adaptive immunity responses. B-cell lymphoma is unique in this context as tumor cells express TLR9 and may harbor latent Epstein-Barr virus (EBV), a gamma-herpesvirus with remarkable oncogenic potential when latent. Latent EBV may be promoted by TLR9 triggering via suppression of lytic EBV. Here, we elaborated an initial assessment of the impact of TLR9 triggering on EBV-positive and EBV-negative B-cell lymphoma using Burkitt''s lymphoma (BL) cell lines as an in vitro model. We show that, independent of the presence of EBV, the TLR9 ligand oligodeoxynucleotide (ODN) CpG-2006 may or may not induce caspase-dependent cell death in BL cells. Moreover, ODN CpG-2006-induced cell death responses of BL cells were associated with TLR9 single-nucleotide polymorphisms (SNPs) rs5743836 or rs352140, which we detected in primary BL tumors and in peripheral blood from healthy individuals at similar frequencies. Thus, our findings suggest that the effect of TLR9 agonists on BL cells should be tested in vitro before installment of therapy and TLR9 SNPs in BL patients should be determined as potential biological markers for the therapeutic response to treatment targeting innate immunity.     

Joint therapy of experimental tumors with diazan in combination with antibiotics]

Antitumor efficiency of combined therapy of experimental tumors with diazan and antibiotics was studied. The experiments were performed with leukosis La and P-388 and Walker carcinosarcoma. The kinetic regularities of the tumor process were used in planning regimens of the combined therapy. It was found that the combined use of diazan with antitumor antibiotics of the anthracyclin series increased the therapy efficacy. Therapeutic synergism of the drugs was noted which was evident from a significant increase in the average life rate and recovery of a part of the animals.     

Effect of hyperthermia in combination with TRAIL on the JNK‐Bim signal transduction pathway and growth of xenograft tumors

Approximately 25% of patients with colorectal cancer develop metastases to the liver, and surgery is currently the best treatment available. But there are several patients who are unresectable, and isolated hepatic perfusion (IHP) offers a different approach in helping to treat these patients. IHP is a method used for isolating the liver and delivering high doses of chemotherapeutic agents. The efficacy of IHP has been improved by combining hyperthermia not only with chemotherapeutics but with other deliverable agents such as tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL). In this study, we used human colorectal cancer CX‐1 cells and treated them with hyperthermia and TRAIL, causing cytotoxicity. We were able to demonstrate that the numbers of live cells were significantly reduced with hyperthermia and 10 ng/ml of TRAIL combined. We also showed that the effect of hyperthermia on TRAIL in our studies was enhancement of the apoptotic pathway by the promotion of JNK and Bim_EL activity as well as PARP cleavage. We have also used our CX‐1 cells to generate tumors in Balb/c nude mice. With intratumoral injections of TRAIL combined with hyperthermia at 42°C, we were able to show a delayed onset of tumor growth in our xenograft model. J. Cell. Biochem. 110: 1073–1081, 2010. Published 2010 Wiley‐Liss, Inc.     

Targeting tumour-initiating cells with TRAIL based combination therapy ensures complete and lasting eradication of multiple myeloma tumours in vivo

Multiple myeloma (MM) remains an incurable disease despite improvements to available treatments and efforts to identify new drug targets. Consequently new approaches are urgently required. We have investigated the potential of native tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), in combination with doxorubicin, to induce apoptotic cell death in phenotypically distinct populations of myeloma cells in vitro and in vivo. The cytotoxic potential of TRAIL alone, and in combination with DOX, was assessed in vitro in purified CD138(+) and CD138(-) cells from the MM cell lines and samples from patients with MM. Mouse xenografts obtained by implanting CD138(-) MM cells were used to assess the efficacy of TRAIL, alone and in combination with DOX, in vivo. CD138(-) cells were shown to be more resistant to the cytotoxic activity of TRAIL than CD138(+) cells and have reduced expression of TRAIL death receptors. This resistance results in preferential killing of CD 138(+) cells during exposure of MM culture to TRAIL. Furthermore, prolonged exposure results in the appearance of TRAIL-resistant CD138(-) cells. However, when TRAIL is combined with doxorubicin, this results in complete eradication of MM cells in vivo. Most importantly, this treatment successfully eliminates CD138(-) cells implicated in tumour initiation and growth maintenance. These findings may explain the failure of current therapies and offer a promising new approach in the quest to cure MM and disseminated cancers.     

Analysis of the neuroinflammatory response to TLR7 stimulation in the brain: comparison of multiple TLR7 and/or TLR8 agonists

Activation of astrocytes and microglia and the production of proinflammatory cytokines and chemokines are often associated with virus infection in the CNS as well as a number of neurological diseases of unknown etiology. These inflammatory responses may be initiated by recognition of pathogen-associated molecular patterns (PAMPs) that stimulate TLRs. TLR7 and TLR8 were identified as eliciting antiviral effects when stimulated by viral ssRNA. In the present study, we examined the potential of TLR7 and/or TLR8 agonists to induce glial activation and neuroinflammation in the CNS by intracerebroventricular inoculation of TLR7 and/or TLR8 agonists in newborn mice. The TLR7 agonist imiquimod induced astrocyte activation and up-regulation of proinflammatory cytokines and chemokines, including IFN-beta, TNF, CCL2, and CXCL10. However, these responses were only of short duration when compared with responses induced by the TLR4 agonist LPS. Interestingly, some of the TLR7 and/or TLR8 agonists differed in their ability to activate glial cells as evidenced by their ability to induce cytokine and chemokine expression both in vivo and in vitro. Thus, TLR7 stimulation can induce neuroinflammatory responses in the brain, but individual TLR7 agonists may differ in their ability to stimulate cells of the CNS.     

Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists,imiquimod and resiquimod

The immune response modifiers, imiquimod and resiquimod, are TLR7 agonists that induce type I interferon in numerous species, including humans. Recently, it was shown that plasmacytoid dendritic cells (pDC) are the primary interferon-producing cells in the blood in response to viral infections. Here, we characterize the activation of human pDC with the TLR7 agonists imiquimod and resiquimod. Results indicate that imiquimod and resiquimod induce IFN-alpha and IFN-omega from purified pDC, and pDC are the principle IFN-producing cells in the blood. Resiquimod-stimulated pDC also produce a number of other cytokines including TNF-alpha and IP-10. Resiquimod enhances co-stimulatory marker expression, CCR7 expression, and pDC viability. Resiquimod was compared throughout the study to the pDC survival factors, IL-3 and IFN-alpha; resiquimod more effectively matures pDC than either IL-3 or IFN-alpha alone. These results demonstrate that imidazoquinoline molecules directly induce pDC maturation as determined by cytokine induction, CCR7 and co-stimulatory marker expression and prolonging viability.     

Deciphering clonality in aneuploid breast tumors using SNP array and sequencing data

Intra-tumor heterogeneity concerns the existence of genetically different subclones within the same tumor. Single sample quantification of heterogeneity relies on precise determination of chromosomal copy numbers throughout the genome, and an assessment of whether identified mutation variant allele fractions match clonal or subclonal copy numbers. We discuss these issues using data from SNP arrays, whole exome sequencing and pathologist purity estimates on several breast cancers characterized by ERBB2 amplification. We show that chromosomal copy numbers can only be estimated from SNP array signals or sequencing depths for subclonal tumor samples with simple subclonal architectures under certain assumptions.

Electronic supplementary material

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