Natural killer cells in cancer : studies on migration and cytotoxicity

The role of natural killer (NK) cells in cancer development has been studied extensively over the last four decades and the increasing knowledge on NK cell regulation has improved both safety and efficacy of treatment. Despite these recent advances the clinical success has to date been modest in treatment of solid tumors, owing both to suboptimal directed migration of NK cells and the tumor cell’s resistance to NK cell-mediated lysis. This thesis focuses on strategies to overcome these critical issues thus improving the anti-tumor effect of adoptive NK cell therapy. In paper I, we have studied the sensitizing effect of doxorubicin on tumor cells to NK cell and T cell-mediated lysis. The potential clinical advantage of using doxorubicin as a preconditioning agent was highlighted in a xenograft mouse model, where mice receiving low-doses of doxorubicin prior to NK cell infusion had a stronger anti-tumor effect of a subsequent NK cell treatment compared to mice receiving only NK cell treatment. Further, we identified TRAIL-signaling as the main pathway responsible for the tumor sensitization due to decreased expression of the anti-apoptotic protein cFLIP. In paper II we have established that the cytotoxicity of NK cells can be augmented by co-culturing them with monocytes in presence of the biphosphonate zoledronic acid (ZA). We observed an upregulated expression of TRAIL on NK cells, through increased levels of monocyte-derived IFNγ in the culture. Thus, NK cells primed with ZA were able to lyse TRAIL-sensitive tumors both in vitro and in vivo. In paper III, we studied CXCL10-mediated migration of NK cells toward solid tumors. We found that ex vivo expansion of NK cells induced a 10-fold increase in CXCR3-receptor expression, which allowed them to migrate towards tumor cells in a CXCL10-dependent manner. In two separate xenograft models we could demonstrate the anti-tumor effect of CXCL10-induced migration of adoptively transferred CXCR3-positive NK cells by their selective targeting of CXCL10-producing tumors, which resulted in reduced tumor progression and prolonged survival. In paper IV, we identified anaplastic thyroid carcinoma (ATC) as a potential novel target for NK cell therapy. We found that ATC cells were sensitive to NKG2D-mediated lysis due to high expression of ULBP2 on tumor cells. In addition, tumor cells produced high levels of CXCL10 which attracted CXCR3-positive NK cells in vitro. In ATC tumor samples we found a suppressed NK cell population although enriched for CXCR3 expression suggesting that CXCL10 may have been involved in the chemoattraction of the NK cells. In conclusion, we have studied some of the important aspects of how NK cells interact with tumor cells and suggested approaches that could improve the use of NK cells in cancer therapy. Moreover, we have identified the highly aggressive tumor ATC as being uniquely sensitive to NK cell lysis and have studied the prospects of developing NK cell therapies for ATC patients

A novel inhibitor of proteasome deubiquitinating activity renders tumor cells sensitive to TRAIL-mediated apoptosis by natural killer cells and T cells

The proteasome inhibitor bortezomib simultaneously renders tumor cells sensitive to killing by natural killer (NK) cells and resistant to killing by tumor-specific T cells. Here, we show that b-AP15, a novel inhibitor of proteasome deubiquitinating activity, sensitizes tumors to both NK and T cell-mediated killing. Exposure to b-AP15 significantly increased the susceptibility of tumor cell lines of various origins to NK (p<0.0002) and T cell (p=0.02) –mediated cytotoxicity. Treatment with b-AP15 resulted in increased TRAIL [tumor necrosis factor-related apoptosis-inducing ligand] receptor-2 expression (p=0.03) and decreased cFLIP expression in tumor cells in vitro. In tumor-bearing SCID/Beige mice, treatment with b-AP15 followed by infusion of either human NK cells or tumor-specific T cells resulted in a significantly delayed tumor progression compared with mice treated with NK cells (p=0.006), T cells (p<0.0001), or b-AP15 alone (p=0.003). Combined infusion of NK and T cells in tumor-bearing BALB/c mice following treatment with b-AP15 resulted in a significantly prolonged long-term survival compared with mice treated with b-AP15 and NK or T cells (p≤0.01). Our findings show that b-AP15-induced sensitization to TRAIL-mediated apoptosis could be used as a novel strategy to augment the anti-cancer effects of adoptively infused NK and T cells in patients with cancer.VetenskapsrådetEuropean Research CouncilAccepte

Kickstarting Immunity in Cold Tumours: Localised Tumour Therapy Combinations With Immune Checkpoint Blockade

Cancer patients with low or absent pre-existing anti-tumour immunity (“cold” tumours) respond poorly to treatment with immune checkpoint inhibitors (ICPI). In order to render these patients susceptible to ICPI, initiation of de novo tumour-targeted immune responses is required. This involves triggering of inflammatory signalling, innate immune activation including recruitment and stimulation of dendritic cells (DCs), and ultimately priming of tumour-specific T cells. The ability of tumour localised therapies to trigger these pathways and act as in situ tumour vaccines is being increasingly explored, with the aspiration of developing combination strategies with ICPI that could generate long-lasting responses. In this effort, it is crucial to consider how therapy-induced changes in the tumour microenvironment (TME) act both as immune stimulants but also, in some cases, exacerbate immune resistance mechanisms. Increasingly refined immune monitoring in pre-clinical studies and analysis of on-treatment biopsies from clinical trials have provided insight into therapy-induced biomarkers of response, as well as actionable targets for optimal synergy between localised therapies and ICB. Here, we review studies on the immunomodulatory effects of novel and experimental localised therapies, as well as the re-evaluation of established therapies, such as radiotherapy, as immune adjuvants with a focus on ICPI combinations

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Genetic and morphological variation in Dunlin Calidris alpina breeding in the Palearctic tundra

The extensive overlap in morphological characters between populations of Dunlin Calidris alpina imposes problems of determining the origin of migrating and wintering birds. The morphology of the birds also varies between the sexes, and the sex of a Dunlin may often be difficult to determine. To clarify if mitochondrial DNA can be used to identify which breeding areas migrating Dunlin come from, we investigated the occurrence of different mtDNA haplotypes in Dunlin from eight breeding areas on the Russian and Siberian tundra. Four haplotypes were found and at most sites more than one haplotype occurred. The European haplotype predominated in the area west of the Taymyr Peninsula, the Siberian haplotype in central Siberia (from the Taymyr Peninsula to the Lopatka Peninsula) and the Beringian haplotype in eastern Siberia. One individual of an Alaskan haplotype, not detected previously among breeding birds outside North America, was found on Wrangel Island. The sex of each bird was identified genetically and the morphology of males and females was analysed separately. Birds with the European haplotype were generally smaller than birds with the Beringian or Alaskan haplotypes. Birds possessing the Siberian haplotype showed intermediate values in most cases. After compensating for differences between sites, males with the Siberian haplotype had significantly longer bills than males having the European haplotype. Multiple regressions indicate that mitochondrial DNA analysis improves models estimating the breeding origin of migrating Dunlin

The impact of surface roughness and permeability in hydroxyapatite bone regeneration membranes

Background One of the crucial aspects in guided bone regeneration is the space maintenance. This is normally created by a membrane, which should primarily be accepted by the surrounding tissues without causing any adverse reactions. The impact of surface topography, biological acceptance as well as permeability of these membranes has been carefully discussed in the literature. Purpose The purpose of this study was to evaluate histologically the bone forming properties inside of hollow hydroxyapatite space-maintaining devices with different inner surfaces and different permeabilities in an animal calvaria model in vivo. Materials and methods A total of 36 hollow domes with three different designs made of hydroxyapatite were surgically attached to the skulls of rabbits. Group 1 had a moderately rough inner surface. Group 2 had a smooth inner surface. Group 3 had the same properties as Group 1 but had macroscopic holes on the top. The domes were left to heal for 12 weeks and were then processed for undecalcified ground sectioning. Histological evaluations were performed using a light microscope and scanning electron microscopy. The bone–implant contact (BIC) percentage along the device was calculated. Results The median percentage of BIC was higher for Group 1 compared with Group 2 (P = 0.004). Group 1 produced a larger median BIC compared with Group 3 (P < 0.0001). Conclusions Within the limits of this preclinical study, these findings suggest that a moderately rough inner surface of a ceramic membrane along with a non-permeable device produces more bone than a smooth inner surface

Guided bone augmentation using ceramic space-maintaining devices : the impact of chemistry.

The purpose of the study was to evaluate histologically, whether vertical bone augmentation can be achieved using a hollow ceramic space maintaining device in a rabbit calvaria model. Furthermore, the chemistry of microporous hydroxyapatite and zirconia were tested to determine which of these two ceramics are most suitable for guided bone generation. 24 hollow domes in two different ceramic materials were placed subperiosteal on rabbit skull bone. The rabbits were sacrificed after 12 weeks and the histology results were analyzed regarding bone-to-material contact and volume of newly formed bone. The results suggest that the effect of the microporous structure of hydroxyapatite seems to facilitate for the bone cells to adhere to the material and that zirconia enhance a slightly larger volume of newly formed bone. In conclusion, the results of the current study demonstrated that ceramic space maintaining devices permits new bone formation and osteoconduction within the dome

Squamous cell carcinoma of the mobile tongue in young adults : A Swedish head &amp; neck cancer register (SweHNCR) population-based analysis of prognosis in relation to age and stage

Increased incidence of squamous cell carcinoma (SCC) of the tongue has been reported in young adults (YA) in several countries since the 1980s and confirmed in later studies. The etiology is unclear, the prognosis has been debated, and conflicting results have been published. Some studies show better survival in young adults than in older patients, some worse, and others no difference. Most studies are based on selected series or include other sites in the oral cavity. The definition of “YA” is arbitrary and varies between studies. It is thus difficult to use in general conclusions. This work uses data from the population-based Swedish Head and Neck Cancer register (SweHNCR), which has > 98% coverage. SweHNCR data includes age, gender, TNM, treatment intention, treatment given, lead times, performance status, and to a lesser degree, smoking habits. The current Swedish population is around 10 million. We analyzed outcomes for 1416 patients diagnosed with SCC of the oral tongue from 2008 to 2017 using 18–39 years to define YA age because it is the range most commonly used. We found no significant difference in relative survival (a proxy for diagnosis-specific survival) between age groups of patients treated with curative intent for SCC of the oral tongue. The stage at time of diagnosis was equally distributed among the age groups. Excess mortality rate correlated mainly with stage, subsite of the tongue, performance status, and lead time to treatment