Cell Kinetic Studies of the Effect of A Combined Therapy With 1-βd-Arabinofuranosylcytosine (Ara-C) and X-Irradiation On the L1210 Ascites Tumour

Abstract. We studied the effect of combined therapy with X-ray and 1-β-D-arabinofuranosylcytosine (Ara-C); firstly the effect of whole-body X-irradiation alone on the proliferation of the L1210 ascites tumour of the mouse was studied by autoradiographic and cytofluorometric (FCM) methods. the effect of X-irradiation with 4 Gy was mainly a cytostatic one leading to an altered distribution of the cells throughout the cycle due to radiation induced mitotic delay. the cytocidal effect is negligible.
As is known from previous studies (Fietkau, Friede & Maurer-Schultze, 1984) the effect of 200 mg/kg Ara-C consists of an inhibition of DNA synthesis and of killing a considerable portion of the L 1210 cells, predominantly of S phase cells.
With respect to the importance for potential therapeutic regimens, the influence of the sequence and the time interval between the two therapeutic steps on the survival of tumour-bearing mice was studied. Most combination therapies significantly increase the survival of tumour-bearing mice compared to the single therapeutic steps; however, no significant differences between the various combined therapies were found. Whole-body X-irradiation with 4 Gy followed by the application of 200 mg/kg Ara-C 10 hr later resulted in the greatest increase of the mean survival time of tumour-bearing animals, from 13.2 to 17.4 days. It was shown that apart from the cytocidal effect on S-phase cells, Ara-C also kills cells sublethally damaged by a preceding X-irradiation.     

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.     

Modulation of radiochemoimmunotherapy-induced B16 melanoma cell death by the pan-caspase inhibitor zVAD-fmk induces anti-tumor immunity in a HMGB1-, nucleotide- and T-cell-dependent manner

One prerequisite that radiotherapy (RT) and chemotherapy (CT) result in anti-tumor immune responses is triggering of immunogenic cell death forms such as necroptosis. The latter is inducible by inhibition of apoptosis with the pan-caspase inhibitor zVAD-fmk. The design of multimodal therapies that overcome melanoma''s resistance to apoptosis is a big challenge of oncoimmunology. As hints exist that immune stimulation by hyperthermia (HT) augments the efficacy of melanoma therapies and that tumors can be sensitized for RT with zVAD-fmk, we asked whether combinations of RT with dacarbazine (DTIC) and/or HT induce immunogenic melanoma cell death and how this is especially influenced by zVAD-fmk. Necroptosis was inducible in poorly immunogenic B16-F10 melanoma cells and zVAD-fmk generally increased melanoma cell necrosis concomitantly with the release of HMGB1. Supernatants (SNs) of melanoma cells whose cell death was modulated with zVAD-fmk induced an upregulation of the activation markers CD86 and MHCII on macrophages. The same was seen on dendritic cells (DCs), but only when zVAD-fmk was added to multimodal tumor treatments including DTIC. DCs of MyD88 KO mice and DCs incubated with SNs containing apyrase did not increase the expression of these activation markers on their surface. The in vivo experiments revealed that zVAD-fmk decreases the tumor growth significantly and results in a significantly reduced tumor infiltration of Tregs when added to multimodal treatment of the tumor with RT, DTIC and HT. Further, a significantly increased DC and CD8+ T-cell infiltration into the tumor and in the draining lymph nodes was induced, as well as an increased expression of IFNγ by CD8+ T cells. However, zVAD-fmk did not further reduce tumor growth in MyD88 KO mice, mice treated with apyrase or RAG KO mice. We conclude that HMGB1, nucleotides and CD8+ T cells mediate zVAD-fmk induced anti-melanoma immune reactions in multimodal therapy settings.The cancer immune editing concept raised by Schreiber and colleagues¹ and the findings that distinct chemotherapeutic agents induce immunogenic cancer cell death forms² opened our minds that standard tumor therapies alone and especially in combination with further immune therapies are capable of inducing anti-tumor immune responses.³ The phenotype of the tumor cells and the tumor microenvironment are altered during therapy and, thereby, the tumor might become visible for the immune system.⁴ A main prerequisite for induction of anti-tumor immunity is triggering of immunogenic tumor cell death forms.⁵Apoptosis is non- or even anti-inflammatory.⁶ In contrast, necrotic cells bear per se a high inflammatory and immunogenic potential. Damage-associated molecular patterns (DAMPs) are released because the plasma membrane of necrotic cells is disturbed.^{7, 8} Danger signals as the high mobility group protein B1 (HMGB1) and the nucleotide adenosine triphosphate (ATP) activate DCs, foster cross-presentation of antigens and consecutively the activation of T cells.⁹ DAMPs therefore link radio- and/or chemotherapy-induced local alterations of the tumor cells and subsequent systemic anti-tumor immune reactions.^{10, 11} HMGB1 is mostly passively released by therapy-induced necrotic tumor cells.¹² The activation of DCs by HMGB1 is induced by its binding to TLR2 or TLR4.^{13, 14} HMGB1 is further required for the migration of maturing DCs.¹⁵ The nucleotide ATP is often actively emitted and acts on purinergic receptors, especially on P2RX7.^{16, 17}Activation of DCs is crucial for the success of multimodal tumor treatments.¹⁸ Several preclinical and clinical studies have demonstrated that tumor cell death induced by radiochemotherapy in combination with intratumoral DC injection induces strong anti-tumor immune responses in several tumor entities.^{19, 20, 21} These responses can be enhanced by hyperthermia (HT). Mild HT is an additive therapy to radiotherapy (RT) and/or chemotherapy (CT) in which tumor tissue is locally heated to temperatures of 40–44 °C for a time period of 1 h. HT fosters protein aggregation and aggravates radiation- and chemotherapy-induced repair of DNA damage.²² In addition, locally applied HT is capable of inducing systemic anti-tumor responses.²³Melanoma is the most dangerous form of skin cancer and its response to CT and RT is poor.²⁴ To overcome melanoma''s resistance to apoptosis, the search for multimodal treatments that aim of inducing immunogenic cell death forms is a big challenge of innovative oncoimmunology,²⁵ as much as to understand the mechanisms of therapy-induced immunogenic melanoma cell death. Nowadays, evidence has come up that necrosis as immunogenic cell death form can also occur in a programmed manner.^{26, 27} Necroptosis is independent of caspases and mainly occurs when caspases are not activated or inhibited.²⁸ The pan-caspase inhibitor zVAD-fmk has been shown to inhibit apoptosis and concomitantly foster necroptosis.²⁹ Further, encouraging preclinical studies have been performed using caspase inhibitors to reduce apoptosis in neurological diseases³⁰ and to reduce angiogenesis in solid tumors.³¹ First hints exist that immune stimulation by HT is capable of augmenting the efficacy of CT and RT treatments in melanoma³² and that solid tumors can be rendered more sensitive to radiation by treatment with the pan-caspase inhibitor zVAD-fmk.³¹ Meaningful data regarding potential clinical efficacy of caspase inhibitors such as zVAD-fmk will only be yielded if the cell death pathways stimulated in model systems reflect that triggered in patients.³³ Therefore, we examined here for the first time whether combinations of the clinically relevant single dose of RT of 2 Gy with the only for metastatic melanoma FDA-approved CT agent dacarbazine (DTIC) or combinations with HT (41.5 °C for 1 h) induce immunogenic melanoma cell death and how zVAD-fmk is capable of improving the melanoma''s immunogenicity by modulating the therapy-induced melanoma cell death.     

Towards a Computable Data Corpus of Temporal Correlations between Drug Administration and Lab Value Changes

Background

The analysis of electronic health records for an automated detection of adverse drug reactions is an approach to solve the problems that arise from traditional methods like spontaneous reporting or manual chart review. Algorithms addressing this task should be modeled on the criteria for a standardized case causality assessment defined by the World Health Organization. One of these criteria is the temporal relationship between drug intake and the occurrence of a reaction or a laboratory test abnormality. Appropriate data that would allow for developing or validating related algorithms is not publicly available, though.

Methods

In order to provide such data, retrospective routine data of drug administrations and temporally corresponding laboratory observations from a university clinic were extracted, transformed and evaluated by experts in terms of a reasonable time relationship between drug administration and lab value alteration.

Result

The result is a data corpus of 400 episodes of normalized laboratory parameter values in temporal context with drug administrations. Each episode has been manually classified whether it contains data that might indicate a temporal correlation between the drug administration and the change of the lab value course, whether such a change is not observable or whether a decision between those two options is not possible due to the data. In addition, each episode has been assigned a concordance value which indicates how difficult it is to assess. This is the first open data corpus of a computable ground truth of temporal correlations between drug administration and lab value alterations.

Discussion

The main purpose of this data corpus is the provision of data for further research and the provision of a ground truth which allows for comparing the outcome of other assessments of this data with the outcome of assessments made by human experts. It can serve as a contribution towards systematic, computerized ADR detection in retrospective data. With this lab value curve data as a basis, algorithms for detecting temporal relationships can be developed, and with the classification made by human experts, these algorithms can immediately be validated. Due to the normalization of the lab value data, it allows for a generic approach rather than for specific or solitary drug/lab value combinations.     

Critical role of spatial interaction between CD8+ and Foxp3+ cells in human gastric cancer: the distance matters

Purpose

In various cancer types, an abundance of FoxP3⁺ regulatory T cells (Treg) has been associated with an unfavorable outcome. Yet, the role of Treg on cancer immunity has been shown to be complex. In single cell marker technique, other tumor-infiltrating lymphocytes (TILs) such as cytotoxic CD8⁺ T cells (CTL) also influenced prognosis. This study for the first time investigates the concurrent spatial distribution pattern of CD8⁺ and FoxP3⁺ TILs and their prognostic impact in human gastric cancer.

Materials and methods

Tumor tissue microarrays of 50 patients with surgically treated adenocarcinoma of the cardia were studied. An immunohistochemical double staining of CD8⁺ and FoxP3⁺ TILs was performed. Cell counts and cell-to-cell distances in tumor epithelium and stroma were evaluated with image-processing software. Metastasis-free survival, no-evidence-of-disease survival, and overall survival were investigated (mean follow-up time 6.9 years).

Results

High intraepithelial infiltration of CD8⁺ and FoxP3⁺ TIL was associated with the improved 10-year metastasis-free survival (83 vs. 54 %, p = 0.04 and 85 vs. 59 %, p = 0.09, respectively). Considering cell-to-cell distance and comparing patients with functional (30–110 μm) versus nonfunctional distances of CD8⁺ and FoxP3⁺ TILs, 10-year survival rates differed between 89 and 55 % (p = 0.009), respectively.

Conclusion

Prognostic influence of tumor-infiltrating immune cells in gastric cancer critically depends on their cell-to-cell distance. FoxP3⁺ TILs must be located within a distance between 30 and 110 μm of CD8⁺ T cells to positively impact on prognosis.     

The immune reaction against allogeneic necrotic cells is reduced in Annexin A5 knock out mice whose macrophages display an anti-inflammatory phenotype

Proteins of the annexin family bind to phospholipids in a Ca²⁺ dependent manner. The exposure of phosphatidylserine (PS) by apoptotic as well as necrotic cells is one major eat-me-signal for macrophages. Annexin A5 (Anx A5) preferentially binds to PS. The availability of Anx A5 knock out (KO) mice allowed us to investigate for the first time if endogenous Anx A5 modulates the immune response towards allogeneic cells. Furthermore, the effect of Anx A5 gene deletion on the phagocytic process as well as on the inflammatory reaction of macrophages was explored. We found that Anx A5 KO mice have a strongly reduced allogeneic cellular immune reaction against primary as well as secondary necrotic cells. In vivo phagocytosis experiments revealed that macrophages of Anx A5 KO mice displayed an increased uptake of necrotic cells. Additionally, an increased secretion of the anti-inflammatory cytokine IL-10 of isolated macrophages of Anx A5 KO mice after contact with necrotic cells was observed. Furthermore, the promoter activity of the Anx A5 gene was enhanced after stimulation of macrophages. The tumour size of an allogeneic tumour regressed faster when endogenous Anx A5 was present. These data demonstrate that endogenous Anx A5 influences the phagocytosis of necrotic cells, modulates the immune response towards allogeneic cells and acts as an inflammatory protein.     

Cell kinetic studies of the effect of a combined therapy with 1-beta D-arabinofuranosylcytosine (Ara-C) and X-irradiation on the L1210 ascites tumour

We studied the effect of combined therapy with X-ray and 1-beta-D-arabinofuranosylcytosine (Ara-C); firstly the effect of whole-body X-irradiation alone on the proliferation of the L1210 ascites tumour of the mouse was studied by autoradiographic and cytofluorometric (FCM) methods. The effect X-irradiation with 4 Gy was mainly a cytostatic one leading to an altered distribution of the cells throughout the cycle due to radiation induced mitotic delay. The cytocidal effect is negligible. As is known from previous studies (Fietkau, Friede & Maurer-Schultze, 1984) the effect of 200 mg/kg Ara-C consists of an inhibition of DNA synthesis and of killing a considerable portion of the L 1210 cells, predominantly of S phase cells. With respect to the importance for potential therapeutic regimens, the influence of the sequence and the time interval between the two therapeutic steps on the survival of tumour-bearing mice was studied. Most combination therapies significantly increase the survival of tumour-bearing mice compared to the single therapeutic steps; however, no significant differences between the various combined therapies were found. Whole-body X-irradiation with 4 Gy followed by the application of 200 mg/kg Ara-C 10 hr later resulted in the greatest increase of the mean survival time of tumour-bearing animals, from 13.2 to 17.4 days. It was shown that apart from the cytocidal effect on S-phase cells, Ara-C also kills cells sublethally damaged by a preceding X-irradiation.