Evaluating a combination treatment of NK cells and reovirus against bladder cancer cells using an in vitro assay to simulate intravesical therapy.

Intravesical treatment using either reovirus or natural killer (NK) cells serves as an efficient strategy for the treatment of bladder cancer cells (BCCs); however, corresponding monotherapies have often shown modest cytotoxicity. The potential of a locoregional combination using high-dose reovirus and NK cell therapy in an intravesical approach has not yet been studied. In this study, we evaluated the effectiveness of reoviruses and expanded NK cells (eNK) as potential strategies for the treatment of bladder cancer. The anti-tumor effects of mono-treatment with reovirus type 3 Dearing strain (RC402 and RP116) and in combination with interleukin (IL)-18/-21-pretreated eNK cells were investigated on BCC lines (5637, HT-1376, and 253J-BV) using intravesical therapy to simulate in vitro model. RP116 and IL-18/-21-pretreated eNK cells exhibited effective cytotoxicity against grade 1 carcinoma (5637 cells) when used alone, but not against HT-1376 (grade 2 carcinoma) and 253J-BV cells (derived from a metastatic site). Notably, combining RP116 with IL-18/-21-pretreated eNK cells displayed effective cytotoxicity against both HT-1376 and 253J-BV cells. Our findings underscore the potential of a combination therapy using reoviruses and NK cells as a promising strategy for treating bladder cancer.

Selective Expansion of NKG2C+ Adaptive NK Cells Using K562 Cells Expressing HLA-E.

Adaptive natural killer (NK) cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs) can be expanded in vivo in response to human cytomegalovirus (HCMV) infection. Developing a method to preferentially expand this subset is essential for effective targeting of allogeneic cancer cells. A previous study developed an in vitro method to generate single KIR+ NK cells for enhanced targeting of the primary acute lymphoblastic leukemia cells; however, the expansion rate was quite low. Here, we present an effective expansion method using genetically modified K562-HLA-E feeder cells for long-term proliferation of adaptive NK cells displaying highly differentiated phenotype and comparable cytotoxicity, CD107a, and interferon-γ (IFN-γ) production. More importantly, our expansion method achieved more than a 10,000-fold expansion of adaptive NK cells after 6 weeks of culture, providing a high yield of alloreactive NK cells for cell therapy against cancer.

Natural Killer Cell Expansion and Cytotoxicity Differ Depending on the Culture Medium Used.

Background: Adoptive cell therapy using umbilical cord blood (UCB)-derived allogeneic natural killer (NK) cells has shown encouraging results. However, because of the insufficient availability of NK cells and limited UCB volume, more effective culture methods are required. NK cell expansion and functionality are largely affected by the culture medium. While human serum is a major affecting component in culture media, the way it regulates NK cell functionality remains elusive. We elucidated the effects of different culture media and human serum supplementation on UCB NK cell expansion and functionality. Methods: UCB NK cells were cultured under stimulation with K562-OX40L-mbIL-18/21 feeder cells and IL-2 and IL-15 in serum-containing and serum-free culture media. The effects of the culture media and human serum supplementation on NK cell expansion and cytotoxicity were evaluated by analyzing the expansion rate, activating and inhibitory receptor levels, and the cytotoxicity of the UCB NK cells. Results: The optimal medium for NK cell expansion was Dulbecco's modified Eagle's medium/Ham's F12 with supplements and that for cytotoxicity was AIM V supplemented with Immune Cell Serum Replacement. Shifting media is an advantageous strategy for obtaining several highly functional UCB NK cells. Live cell imaging and killing time measurement revealed that human serum enhanced NK cell proliferation but delayed target recognition, resulting in reduced cytotoxicity. Conclusions: Culture medium supplementation with human serum strongly affects UCB NK cell expansion and functionality. Thus, culture media should be carefully selected to ensure both NK cell quantity and quality for adoptive cell therapy.

High-Throughput 3D In Vitro Tumor Vasculature Model for Real-Time Monitoring of Immune Cell Infiltration and Cytotoxicity.

Recent advances in anticancer therapy have shown dramatic improvements in clinical outcomes, and adoptive cell therapy has emerged as a type of immunotherapy that can modulate immune responses by transferring engineered immune cells. However, a small percentage of responders and their toxicity remain as challenges. Three-dimensional (3D) in vitro models of the tumor microenvironment (TME) have the potential to provide a platform for assessing and predicting responses to therapy. This paper describes an in vitro 3D tumor model that incorporates clusters of colorectal cancer (CRC) cells around perfusable vascular networks to validate immune-cell-mediated cytotoxicity against cancer cells. The platform is based on an injection-molded 3D co-culture model and composed of 28 microwells where separate identical vascularized cancer models can be formed. It allows robust hydrogel patterning for 3D culture that enables high-throughput experimentation. The uniformity of the devices resulted in reproducible experiments that allowed 10× more experiments to be performed when compared to conventional polydimethylsiloxane (PDMS)-based microfluidic devices. To demonstrate its capability, primary natural killer (NK) cells were introduced into the vascularized tumor network, and their activities were monitored using live-cell imaging. Extravasation, migration, and cytotoxic activity against six types of CRC cell lines were tested and compared. The consensus molecular subtypes (CMS) of CRC with distinct immune responses resulted in the highest NK cell cytotoxicity against CMS1 cancer cells. These results show the potential of our vascularized tumor model for understanding various steps involved in the immune response for the assessment of adoptive cell therapy.

The significance of amplitude and duration of fetal heart rate acceleration in non-stress test analysis.

OBJECTIVE: This study was conducted to assess the relative significance of the amplitude versus the duration of accelerations in non-stress test (NST) analysis. MATERIALS AND METHODS: A total of 3055 normal fetal heart rate (FHR) tracings at 30-42 weeks' gestation were analyzed by automated FHR analyzing software. Accelerations were classified as one of four combinations of amplitude and duration: 15 bpm-15 seconds (Acc15-15), 15 bpm-10 seconds (Acc15-10), 10 bpm-15 seconds (Acc10-15) and 10 bpm-10 seconds (Acc10-10). We estimated the correlation among the FHR acceleration combinations using correlation analysis based on linear regression models. RESULTS: Linear regression models demonstrated statistically significant linear associations between Acc15-15 and Acc15-10 (r(2) = 0.998, p < 0.0001) and between Acc10-10 and Acc10-15 (r(2) = 0.989, p < 0.0001). There was significant association based on amplitude and relatively low correlation based on duration (Pearson correlation coefficient = 0.99 between Acc10-10 and Acc10-15, and 0.99 between Acc15-15 and Acc15-10). In the relationships of the FHR-work values, amplitude was a more important component of FHR acceleration than duration [Acc10-10 (1.67 beat) < Acc10-15 (2.50 beats) = Acc15-10 (2.50 beats) < Acc15-15 (3.75 beats)]. CONCLUSION: Amplitude was a more significant component of FHR acceleration than duration in the computerized analysis of NST.

Conventional linear criteria with sample entropy in the interpretation of reactive antepartum fetal heart rate tracings.

OBJECTIVE: To compare the test duration times and rate of nonreactive results between the conventional linear reactive criteria (CLRC) and the modified nonlinear reactive criteria (MNRC) in electronic fetal heart rate (FHR) monitoring. The MNRC are the CLRC with the addition of approximate entropy or sample entropy. METHODS: One thousand women with singleton pregnancies between 30 and 37 weeks' gestation were selected. They visited Hanyang University Hospital for non-stress tests (NSTs). In these patients, FHR tracings were recorded for 50 min including the first and second 20 min of NST and 10 min of vibroacoustic stimulation test. All patients had a reactive NST with CLRC in these antepartum FHR tests. The rate of nonreactive tests and test duration times of these linear and nonlinear criteria were calculated and compared. RESULTS: The rate of nonreactive tests within a 20- and 40-min period with the MNRC (6.8 and 1.2%) was significantly lower than with the CLRC (35.2 and 10.5%, respectively; p < 0.001). To meet the reactive criteria, the mean durations of the MNRC and the CLRC were 11.47 (95% CI, 11.11-11.84) and 18.45 (95% CI, 17.79-19.12) min, respectively (p < 0.001). The MNRC led to a significant reduction in testing time of 37.8% compared to the CLRC. The rate of reactive tests using only sample entropy (0.20), calculated over 5-min segments of FHR tracings, was 88.3% (95% CI, 88.0-88.7). It was close to the results using the MNRC (93.2%) in the 20-min time window. CONCLUSION: The use of the MNRC significantly reduced the test duration times and decreased the rate of nonreactive tests when compared to the CLRC. It may be useful in providing more rapid and accurate assessment of the fetus in electronic FHR monitoring.

Difference of fetal heart rate accelerations based on 10 and 15 beats per minute.

AIM: To evaluate the correlation of 10 b.p.m.-10 s and 15 b.p.m.-15 s fetal heart rate (FHR) accelerations in gestations before or after 32 weeks using computerized analysis. METHODS: A combination of amplitude and duration of FHR accelerations, 10 b.p.m.-10 s (Acc10-10) and 15 b.p.m.-15 s (Acc15-15), was analyzed according to gestational weeks between January 1999 and December 2005 in 2358 normal pregnant women who received a non-stress test at 30-42 gestational weeks. A linear regression model between Acc10-10 and Acc15-15 was estimated, and the duration difference between 10 b.p.m. and 15 b.p.m. was converted into seconds. RESULTS: Before 32 weeks of gestation, the mean number of FHR accelerations based on 15 b.p.m. was below 2.00 (mean + or - standard error, 1.58 + or - 0.19). The correlation between Acc10-10 and Acc15-15 was Acc15-15 + 2.8 = Acc10-10 (r(2) = 0.94, P = 0.0013). The mean duration difference between 10 b.p.m. and 15 b.p.m. was 36.8 s (range, 4-227 s). CONCLUSIONS: Our study verified the difference of Acc10-10 and Acc15-15 using computerized analysis as the base of visual interpretation of the definition of FHR acceleration. Acc15-15 did not occur often enough to be relevant to the definition of FHR acceleration before 32 weeks' gestation. The difference between the mean number of FHR accelerations based on 10 b.p.m. and 15 b.p.m. within a 20-min window was 2.8.

Determination of fetal heart rate reactivity from a single 20-min window of non-stress testing in compromised fetuses.

AIMS: To shorten the analysis time needed for non-stress testing (NST) without decreasing efficacy in compromised fetuses. METHODS: We selected 80 cases with a 5-min Apgar score <7 as a study group and 259 cases with a 5-min Apgar score >/=9 as a control group. We applied four different criteria (A, B, C, and D) to each study and control group for the first 20-min window of NST data to evaluate reactivity. Criteria A, B, and C consisted of conventional reactivity criteria according to amplitude (15 or 10 beats per minute), duration (15 or 10 s) and weeks of gestation (/=32), and criteria D combined criteria C with approximate entropy (ApEn). RESULTS: The sensitivity of criteria D (91.25%) was greater than the other three criteria (P<0.0001). The specificities of criteria C (96.14%) and D (99.23%) were also higher than criteria A and B (P<0.0001). The positive and negative predictive value of criteria D were better than that of criteria C (97.33 vs. 83.87, P=0.0066) and (97.35 vs. 89.89, P=0.0004), respectively. CONCLUSION: Adding ApEn to the conventional criteria for reactivity shortened NST analysis time without decreasing efficacy, facilitating a decision of reactivity within a single 20-min NST window.

Ex vivo expansion and clonality of CD34+ selected cells from bone marrow and cord blood in a serum-free media.

Although umbilical cord blood is increasingly being used in allogeneic marrow transplantation, delayed platelet engraftment is often a concern for cord blood transplant recipients. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from a bone marrow source, and cord blood, in a serum-free Media. The CD34+ cells, selected from bone marrow (BM) and umbilical cord blood (CB), were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at days 0, 4, 7, and 14 under the combination of growth factors, with cell counts. The cytokines included the recombinant human megakaryocyte growth and development (100 ng/ml), interleukin-3 (10 ng/ml), stem cell factor (100 ng/ml), flt-3 ligand (50 ng/ml) and interleukin-11 (200 ng/ml). The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the BM at day 7 (3.0 fold increase than BM), day 14 (2.4 fold), and day 17 (2.6 fold). The colony count of the BFU-E/CFU-E per CD34+ cell at day 0 was 0.14 +/- 0.023 in the CB, which was significantly higher than 0.071 +/- 0.015 in the BM. The CB-selected CD34+ cells produced more BFU-E colonies than the BM on culture days 4, 7, and 14. The BFU-E colonies from the CB cells increased markedly on culture days 4 and 7, with a 4-fold increase at day 14. The colony count of the CFU-Mk per CD34+ cell at day 0 was 0.047 +/- 0.011 in the CB-selected CD34+ cells cultures, which was higher than the 0.026 +/- 0.014 in the BM. The CB-selected CD34+ cells produced more CFU-Mk colonies than the BM on culture days 4, 7 and 14. In conclusion, the ex vivo expansion of the CB cells may be very promising in producing total cellular expansion, CFU-Mk and BFU-E compared with BM, especially at day 7. The ex vivo expansion of the CB may have rationale in making an ex vivo culture for 7 to 14 d.