Expert answer:one-way ANOVA calculator


Solved by verified expert:I need to make a presentation in class (research methods) using online calculator: one-way ANOVA

Unformatted Attachment Preview

Don't use plagiarized sources. Get Your Custom Essay on
Expert answer:one-way ANOVA calculator
Just from $10/Page
Order Essay

published: 09 April 2019
doi: 10.3389/fimmu.2019.00496
Defective Localization With Impaired
Tumor Cytotoxicity Contributes to
the Immune Escape of NK Cells in
Pancreatic Cancer Patients
Seon Ah Lim 1† , Jungwon Kim 1† , Seunghyun Jeon 1† , Min Hwa Shin 1 , Joonha Kwon 1 ,
Tae-Jin Kim 1 , Kyungtaek Im 1 , Youngmin Han 2 , Wooil Kwon 2 , Sun-Whe Kim 2 ,
Cassian Yee 3 , Seong-Jin Kim 4*, Jin-Young Jang 2* and Kyung-Mi Lee 1,3,5*
Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul, South Korea,
Department of Surgery and Cancer Research Institute, Seoul National University Hospital, Seoul National University College
of Medicine, Seoul, South Korea, 3 Department of Melanoma Medical Oncology and Immunology, MD Anderson Cancer
Center, Houston, TX, United States, 4 Precision Medicine Research Center, Advanced Institutes of Convergence Technology,
Seoul National University, Suwon, South Korea, 5 Center for Bio- Integrated Electronics, Simpson Querrey Institute, Evanston,
IL, United States
Edited by:
Andreas Pircher,
Innsbruck Medical University, Austria
Reviewed by:
Sieghart Sopper,
Innsbruck Medical University, Austria
Alessandro Poggi,
Ospedale Policlinico San Martino, Italy
Franz Rödel,
Universitätsklinikum Frankfurt,
Seong-Jin Kim
Jin-Young Jang
Kyung-Mi Lee
† These
authors have contributed
equally to this work
Specialty section:
This article was submitted to
Cancer Immunity and Immunotherapy,
a section of the journal
Frontiers in Immunology
Received: 16 October 2018
Accepted: 25 February 2019
Published: 09 April 2019
Lim SA, Kim J, Jeon S, Shin MH,
Kwon J, Kim T-J, Im K, Han Y,
Kwon W, Kim S-W, Yee C, Kim S-J,
Jang J-Y and Lee K-M (2019)
Defective Localization With Impaired
Tumor Cytotoxicity Contributes to the
Immune Escape of NK Cells in
Pancreatic Cancer Patients.
Front. Immunol. 10:496.
doi: 10.3389/fimmu.2019.00496
Tumor-infiltrating lymphocytes (TILs), found in patients with advanced pancreatic ductal
adenocarcinoma (PDAC), are shown to correlate with overall survival (OS) rate. Although
majority of TILs consist of CD8+ /CD4+ T cells, the presence of NK cells and their role
in the pathogenesis of PDAC remains elusive. We performed comprehensive analyses
of TIL, PBMC, and autologous tumor cells from 80 enrolled resectable PDAC patients
to comprehend the NK cell defects within PDAC. Extremely low frequencies of NK
cells (<0.5%) were found within PDAC tumors, which was attributable not to the low expression of tumor chemokines, but to the lack of chemokine receptor, CXCR2. Forced expression of CXCR2 in patients’ NK cells rendered them capable of trafficking into PDAC. Furthermore, NK cells exhibited impaired cell-mediated killing of autologous PDAC cells, primarily due to insufficient ligation of NKG2D and DNAM-1, and failed to proliferate within the hypoxic tumor microenvironment. Importantly, these defects could be overcome by ex-vivo stimulation of NK cells from such patients. Importantly, when the proliferative capacity of NK cells in vitro was used to stratify patients on the basis of cell expansion, patients whose NK cells proliferated <250-fold experienced significantly lower DFS and OS than those with ≥250-fold. Ex-vivo activation of NK cells restored tumor trafficking and reactivity, hence provided a therapeutic modality while their fold expansion could be a potentially significant prognostic indicator of OS and DFS in such patients. Keywords: pancreatic cancer, cancer immunobiology, cellular immunology, immunotherapy, chemokines INTRODUCTION Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies (1, 2). Majority of patients with PDAC are diagnosed with distant metastasis or locally advanced stages, rendering them surgically inoperable at the time of diagnosis (3). Although patients with metastases from colorectal or breast cancer have reported improved outcomes with the advent of new chemotherapeutic agents over the last decade, there has not been much increase in survival of Frontiers in Immunology | 1 April 2019 | Volume 10 | Article 496 Lim et al. NK Cells Impairment in Pancreatic Cancer patients with pancreatic cancer. Anatomical location, delayed diagnosis, and chemo-resistance, associated with a stromal compartment that serves as a barrier for chemotherapeutic drugs, are key factors contributing to the poor prognosis observed in patients with PDAC (4). Furthermore, PDAC has been shown to be inherently immunosuppressive, producing TGF-β, IL-10, IDO, and MMPs (5–9), thereby allowing their escape from immune surveillance. For example, the PDAC tumor microenvironment favors the expression of co-inhibitory ligands, PDL-1 and PDL-2, and inhibits HLA class I expression in tumor cells (10–12) while promoting the emergence of regulatory T cells (Treg) and tumor-associated macrophages (TAM) that secrete immunosuppressive cytokines (5, 13). Moreover, pancreatic carcinoma are supplied with insufficient and aberrant blood vessels, and are hence extremely hypoxic. Under low oxygen conditions, growth of stellate cells, the major fibroblastic cells of the pancreas co-residing with tumor cells, is significantly accelerated (14). Despite the severe desmoplastic and immunosuppressive nature of PDAC microenvironment, a significant level of Tumorinfiltrating lymphocytes (TIL) infiltration has been found in patients with PDAC that correlates with clinical prognosis (15, 16). A high level of CD8+ and CD4+ T cells, together with a low number of Tregs, was found to be associated with improved survival in such patients (17). However, most of the activated T cells, localized to PDAC, express an array of co-inhibitory receptors, including PD-1, LAG-3, TIM3, and CTLA-4, which can induce tolerance and exhaustion of Antigen-specific T cells after the initial recognition of tumors (9, 18–21). NK cells are bone marrow-derived large granular lymphocytes shown to provide the body’s first line of defense (22, 23). NK cell lytic functions are regulated by an array of activating and inhibitory receptors on the cell surface, leading to the release of cytotoxic granules containing perforin and granzymes (24). Activating receptors in charge of tumor cytolysis in NK cells include NKG2D (Natural killer group2, member D), DNAM1(DNAX accessory molecule-1), and NCR (Natural Cytotoxicity Receptors, NKp30, NKp44, NKp46) while KIR (Killer inhibitory receptors) and CD94-NKG2 heterodimers serve as inhibitory receptors (25, 26). It has been shown previously that progression of pancreatic cancer is closely associated with dysfunctional circulating NK cells in PBMC (27). However, the presence of NK cells within pancreatic tumors and their role in the progression of PDAC remains unclear. To address these issues, we obtained tumor and peripheral blood samples from such patients undergoing surgical resection, and performed a detailed analysis of NK cell frequency and their proliferation profile. Furthermore, we isolated tumor cells from the patients to assess anti-tumor function of the NK cells in autologous settings, and attempted to correlate these parameters with clinically annotated findings. TABLE 1 | Patients’ characteristics and TNM staging of pancreatic cancers. Characteristics % GENDER Male 49 61.3 Female 31 38.7 AGE ≤65 30 37.5 >65
Head of pancreas
Body of pancreas
Tail of pancreas
I (A/B)
II (A/B)
Korean Cell Line Bank, Seoul, Korea) were cultured in
RPMI 1640 medium supplemented with 10% FBS, 100 U/ml
penicillin, and 100 U/ml streptomycin. PANC-1, MIA PaCa-2
(human pancreatic cancer cell line; ATCC), and Hep3B (human
hepatocellular carcinoma cell line; ATCC) were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin.
Human PBMCs were isolated from healthy donors and
patients with pancreatic cancer. The Epstein-Barr virus (EBV)transformed lymphoblastoid B cell line (LCL) was derived from
PBMCs with EBV supernatant of B95-8 cells. For hypoxic
treatments, cells were cultured in an anaerobic incubator
(VISION CO2 O2 INCUBATOR, Vision Scientific Co., Ltd.).
All studies containing human subjects were approved by the
with donor’s consent (1040548-KU-IRB-16-103-A-2).
Patient Characteristics
A total of 80 patients were enrolled from May 1, 2015 to
July 31, 2016. Patient profiles describing age, sex, percentage of
neoadjuvant therapy, primary tumor location, and TNM stage
are listed in Table 1.
Preparation of Primary PDAC Tumor Cells
and Isolation of TILs From Patients
Undergoing Resection Surgery
Tumor specimens from 80 patients with PDAC were obtained
from the Department of Surgery, SEOUL NATIONAL
UNIVERSITY HOSPITAL (SNUH) and processed according
to the guidelines provided by the ethics committee of SNUH
(1503-093-657). Pathological features of all tissues were assessed
according to WHO classification and AJCC staging (7th edition).
Cell Lines and Cell Culture
K562 (human erythroblastoid cell line; ATCC, Manassas, VA)
and Jurkat, indicated as KL-1 (human T lymphoblast line;
Frontiers in Immunology |
April 2019 | Volume 10 | Article 496
Lim et al.
NK Cells Impairment in Pancreatic Cancer
Cells were co-cultured at the indicated effector-to-target (E:T)
ratios and incubated at 37◦ C for 4 h. After incubation, 80 µl of
the supernatant was harvested and transferred to a new plate.
Samples were measured using a Hidex Sense microplate reader
(Ex: 485 nm/Em: 530 nm). Percent lysis was calculated with the
same formula used for the 51 Cr release assay.
The surgically dissected pancreatic tumor masses from patients
with PDAC were washed with Phosphate-buffered saline (PBS)
and minced into 3–5 mm2 slices, and collected in RPMI 1640
supplemented with 10% FBS, 100 U/ml penicillin, and 100 U/ml
streptomycin. Sliced tumor fragments were dissociated with
750 U/ml of type IV collagenase (Worthington Biochemical
Corporation, Lakewood, NJ), and incubated for 1 h at 37◦ C
in 5% CO2 , with rotation. After washing twice with RPMI
1640, a part of the cells was used for CD45 positive selection
using magnetic-activated cell sorting, and the rest was used for
culturing primary pancreatic cancer cells.
Quantitative Real-Time Polymerase Chain
Reaction (qRT-PCR) Analysis
Total RNA was extracted from the cells and tissue specimens
using TRIzol reagent (Life Technologies, Carlsbad, CA)
according to the manufacturer’s instructions. All quantitative
real-time PCR amplifications were performed using StepOnePlus
(Applied Biosystems, Foster City, CA) and SYBR green supermix
(Bio-Rad). Gene expression was normalized using 18S rRNA.
Primer sequences used are listed in Table S1.
Antibodies and Flow Cytometry
Anti-human CD2 FITC (RPA-2.10), CD8 APC (OKT8), CCR7
PerCP-Cy5.5 (3D12), CD57 PE (TB01), 2B4 PE (DM244), CD25
PE (BC96), Foxp3 PerCP-Cy5.5 (PCH101), LAG-3 PerCP-Cy5.5
(3DS223H), CD160 PE (BY55), CXCR4 APC (12G5), ICAM-1 PE
(HA58), CD11a FITC (HI111), NKG2D APC (5C6), and CD69
FITC (FN50) mAbs were purchased from eBioscience. Antihuman CXCR3 PE (1C6), CCR10 PerCP-Cy5.5 (1B5), DNAM1 FITC (DX11), CD94 FITC (HP-3D9), CD158a PE (HP-3E4),
KIR-NKAT2 FITC (DX27), CD20 PerCP (L27), NKp46 APC
(9E2), CD158b PE (CH-L), CD132 PE (AG184), NKp30 PE
(P30-15), NKB1 FITC (CX9), and CD122 PE (Mik-b3) mAbs
were purchased from BD Pharmingen. Anti-human CD3 FITC
(OKT3), KLRG1 FITC (2F1), CD56 PE (HCD56), TIM3 PE (F382E2), CCR5 FITC (HEK/1/85a), CCR8 PE (L263G8), CD16 FITC
(3G8), CXCR6 APC (K04125), CXCR2 PerCP-Cy5.5 (5E8), and
CD45 Alexa700 (HI30) mAbs were purchased from BioLegend.
Anti-human NKG2A PerCP (131411), NKG2C APC (134591)
mAbs were purchased from R&D Systems. Purified anti-human
DNAM-1 (102511), NKp30 (210845), NKp44 (253415), NKp46
(195314), and NKG2D (149810) from R&D Systems and ICAM1 (Clone R6-5-D6) from Bio X Cell were used to inhibit the
binding of receptors to their ligands. For surface staining, cells
were stained with the indicated FITC-, APC-, PE-, or PerCPconjugated mAbs in 0.1 ml FACS buffer (BD Biosciences). Flow
cytometry was performed with FACSCanto II (BD Biosciences,
San Diego, CA) and data were analyzed with FlowJo (Tree Star,
Ashland, OR) software.
Lentiviral Transduction of CXCR2 in NK
Full-length human CXCR2 was obtained from Addgene (cat
#66260, MA) and amplified by polymerase chain reaction.
pCDH-521A lentiviral plasmids carrying CXCR2 were cotransfected with packaging plasmids into 293 FT cells using
CaCl2 , and supernatant was collected after 72 h of culture.
A total of 5 × 106 NK cells were seeded with virus in
each well of 48-well plates with complete medium containing
8 µg/ml Polybrene (hexadimethrine bromide, Sigma-Aldrich).
The following day, viral supernatant was removed and replaced
with growth medium. Four days later, transduced NK cells were
collected for further analysis.
Migration Assay
Vector or CXCR2-transduced NK cells (1.5 × 105 cells) were
placed in 0.2 ml of complete medium in the upper chamber (5.0µm pore) of a 24-well Transwell plate (Costar). Medium (1 mL)
containing MIA PaCa-2 or primary tumor cells were placed in
the lower chamber, and the plates were incubated for 4 h at
37◦ C. The number of cells in the lower chamber was manually
counted. The percentage of CD3-FITC negative and CD56-APC
positive NK cells in each well was quantified by FACSCanto II
(BD Biosciences), and analyzed with FlowJo software (Tree Star).
Cytotoxicity Assay
Expansion of NK Cells
51 Cr-release
assay was conducted according to the published
protocol (28). Briefly, target cells were labeled with 51 Cr (Perkin
Elmer, Boston, MA) at 50 µCi/5 × 105 cells and incubated for
4 h at 5 × 103 cells/well with serial dilution of PBMCs. In some
experiments, the assay was performed in presence of 5 µg/ml
of blocking anti-NKG2D, anti-DNAM-1, anti-NKp30, antiNKp44, anti-NKp46, or anti-ICAM1 mAbs or isotype controls.
The γ-scintillation of supernatant was quantified using a γcounter (Perkin Elmer). Percentage of specific lysis was measured
with the following formula: 100 × (experimental releasespontaneous release)/(maximum release-spontaneous release).
NK cell cytotoxicity, in hypoxic condition, was determined using
the Calcein-AM release assay. Target cells were labeled with
2–4 µg/ml (titrated for each tumor cell line) of Calcein-AM
(Sigma-Aldrich) for 30 min at 37◦ C with occasional shaking.
Frontiers in Immunology |
Expansion of NK cells was performed as described previously
(29). Briefly, PBMCs, prepared from peripheral blood using
Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden), were cocultured with gamma-irradiated (100 Gy) KL-1 and LCL feeder
cell lines in RPMI 1640 medium supplemented with 10% FBS and
recombinant human IL-2 (500 U/ml; rhIL-2, Proleukin; Novartis,
Basel, Switzerland). Medium was changed every 3 days up to 6
days, and every 4 days up to 18 days, thereafter. Fresh IL-2 was
added when medium was changed during the culture. On day
6, expanded NK cells were transferred to T25 or T75 flasks at
a concentration of 0.25 × 106 cells/ml. The absolute number of
NK cells was calculated by multiplying the total number of viable
cells by the percentage of CD56+ CD3− cells, measured by flow
cytometry. Fold change was determined by dividing the number
April 2019 | Volume 10 | Article 496
Lim et al.
NK Cells Impairment in Pancreatic Cancer
HD controls while over 50% of reduction of B, CD4 T, and CD8 T
cells were reduced in the patients (Figure 1B, bottom).
In contrast, significantly high percentages of CD4+ CD3+
(42.76 ± 11.26%) and CD8+ CD3+ (39.30 ± 1.10%) T cells were
found in TILs, as compared to those in PBMCs CD4+ CD3+
(31.64 ± 12.43%) and CD8+ CD3+ (18.85 ± 9.81%), indicating
active localization of T cells to pancreatic tumors (17). The
percentage of CD20+ B cells in TILs (3.65 ± 4.80%) was
similar to that in PBMCs (5.58 ± 3.75%), but the percentage
of CD4 Treg was 5–6 fold elevated in TILs, implying signs of
immunosuppressive microenvironment. These data demonstrate
that NK cells exhibit severely impaired tumor localization,
distinct from other types of lymphocytes shown in patients with
resectable pancreatic tumor.
of viable NK cells present at the designated day of culture by the
number of viable NK cells at the beginning of culture.
In vivo Tumor Challenges
Six to 9-week-old female NOD scid gamma (NSG) mice
were purchased from Jackson laboratories, and maintained
at Korea University (Seoul, Korea) animal facilities under
specific pathogen-free conditions. All animal experiments
were performed in accordance with national and institutional
guidelines (KOREA-2017-0066-C1). Approximately, 1 × 107
MIA PaCa-2 cells were subcutaneously injected into the right
flank of NSG mice, followed by intravenous injection of 1 ×
107 expanded NK cells, 10 days later, at days 7, 14, 21, 28, 35,
42, and 49. Tumor volumes were measured for up to 50 days
following immunization.
Surface Expression of CXCR2 Chemokine
Receptor Is Reduced in Circulating NK
Cells of Patients With PDAC
Statistical analysis was performed using SPSS version 23.0
(IBM, Armonk, NY). Nominal and continuous variables were
compared using the χ2 tests and Student’s t test, respectively.
Survival rates were calculated using the Kaplan-Meier method,
and the log-rank test was used to analyze the differences. The
survival time and disease-free time were calculated from the
start of surgery. Variables that were statistically significant in
univariate analysis were included in multivariate analysis using
the Cox proportional hazards regression. Two-sided p values
of <0.05 were considered significant. A two-tailed Student’s ttest was used for statistical comparison of two groups, where indicated, and p-values (∗ p ≤ 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001) were taken as statistically significant. Based on the speculation that the low frequency of NK cells in TILs of patients with PDAC might be due to impaired chemokine secretion associated with NK cell trafficking, we analyzed the expression of chemokine ligands, produced by the pancreatic tumors, by quantitative real-time qPCR (Figure 2A, Table S1). Non-tumor tissues harvested from the same patients were used as controls. While non-tumor tissues did not show any significant level of CXCL chemokines, tumor tissues expressed high levels of CXCL3 and CXCL5 and relatively low levels of CXCL1, CXCL2, and CXCL7, similar to those reported previously (30–34). Expression of CXCL6 and CXCL8 was minimal, compared to other CXCL ligands. Furthermore, PDAC tumors did not express CXCL12, a ligand for CXCR4. These data demonstrate that lymphocytes expressing CXCR2 can efficiently localize toward pancreatic tumors. Contrary to those from HD, NK cells from patients with PDAC showed reduced surface expression of CXCR2 (Figure 2B). Downregulation of surface CXCR2 on NK cells was apparent on CD56low NK cell populations (Figure 2C, Figure S5), but was not associated with the reduced CXCR2 mRNA expression (Figure S5) or reduced the number of CD56low populations in the patients (Figure 2C). These data suggest that downregulation of CXCR2 on the cell surface of NK cells in PDAC patients is likely to occur at the post-transcription level or post-translational involving protein internalization or degradation pathways associated with immunosuppression (35–37). It is noteworthy that the surface expression of CXCR4 on NK cells was comparable between PDAC and HD (Figure 2B), and its ligand CXCL12 was not expr ... Purchase answer to see full attachment

Place your order
(550 words)

Approximate price: $22

Calculate the price of your order

550 words
We'll send you the first draft for approval by September 11, 2018 at 10:52 AM
Total price:
The price is based on these factors:
Academic level
Number of pages
Basic features
  • Free title page and bibliography
  • Unlimited revisions
  • Plagiarism-free guarantee
  • Money-back guarantee
  • 24/7 support
On-demand options
  • Writer’s samples
  • Part-by-part delivery
  • Overnight delivery
  • Copies of used sources
  • Expert Proofreading
Paper format
  • 275 words per page
  • 12 pt Arial/Times New Roman
  • Double line spacing
  • Any citation style (APA, MLA, Chicago/Turabian, Harvard)

Our guarantees

Delivering a high-quality product at a reasonable price is not enough anymore.
That’s why we have developed 5 beneficial guarantees that will make your experience with our service enjoyable, easy, and safe.

Money-back guarantee

You have to be 100% sure of the quality of your product to give a money-back guarantee. This describes us perfectly. Make sure that this guarantee is totally transparent.

Read more

Zero-plagiarism guarantee

Each paper is composed from scratch, according to your instructions. It is then checked by our plagiarism-detection software. There is no gap where plagiarism could squeeze in.

Read more

Free-revision policy

Thanks to our free revisions, there is no way for you to be unsatisfied. We will work on your paper until you are completely happy with the result.

Read more

Privacy policy

Your email is safe, as we store it according to international data protection rules. Your bank details are secure, as we use only reliable payment systems.

Read more

Fair-cooperation guarantee

By sending us your money, you buy the service we provide. Check out our terms and conditions if you prefer business talks to be laid out in official language.

Read more

Order your essay today and save 30% with the discount code ESSAYSHELP