table_3_Combination of Cytokine-Induced Killer Cells and Programmed Cell Death-1 Blockade Works Synergistically to Enhance Therapeutic Efficacy in Metastatic Renal Cell Carcinoma and Non-Small Cell Lung Cancer.PDF

Introduction<p>Programmed cell death-1 (PD-1) inhibition therapy has changed the treatment paradigm of metastatic renal cell carcinoma (MRCC) and non-small cell lung cancer (NSCLC). However, attempts to use the drug as a single agent have achieved only limited clinical success. To further enhance the clinical benefits of monotherapy, combination therapies will likely be necessary. Cytokine-induced killer (CIK) cells are a heterogeneous subset of ex vivo expanded T lymphocytes that have been shown to prolong the survival of cancer patients. We are conducting a study to evaluate the efficacy of PD-1 inhibitor in combination with CIK cells in relapsed/refractory MRCC and NSCLC and to analyze potential biomarkers to predict which patients will benefit most from the combined therapy.</p>Case presentation<p>The results of two patients treated in an ongoing clinical trial for MRCC and NSCLC are described here. The tumor biopsy from Patient 1 exhibited moderate CD3⁺ T cell infiltration, but no PD-1 or PD-L1 expression. The tumor cells from Patient 2 strongly expressed PD-L1, and there was extensive tumor infiltration by CD3⁺ T cells; however, no PD-1 staining was seen. Non-synonymous single nucleotide variant (nsSNVs), along with higher indel mutations, in Patient 1 and nsSNVs along with higher tumor mutation burden in Patient 2 correlate with tumor-infiltrating CD3⁺ lymphocyte density. Patient 1 achieved a complete response, and Patient 2 achieved a near-complete response.</p>Conclusion<p>A PD-1 inhibitor in combination with CIK cells led to potent antitumor activity in MRCC and NSCLC; CD3⁺ T cell infiltration in baseline tumor biopsies is a potential predictive biomarker. This approach is being further investigated in an ongoing phase I trial.</p

Skeleton-Based 3D Object Retrieval Using Retina-Like Feature Descriptor

Skeleton-based 3D object retrieval is a very efficient method to query the sketch databases in numerous applications. However, few skeleton images are found so far in existing sketch benchmarks. In this paper, we provide an initial benchmark dataset consisting of skeleton sketches, including hand-drawn skeletons and skeletons extracted from 3D objects, and both of them are used to form a generic object class. Then we present a method for skeleton-based 3D object retrieval using a retina-like feature descriptor (S3DOR-RFD) based on the structural property of the human retina for processing complex visual information in a very efficient way. As part of the S3DOR-RFD algorithm, we combine artificial bee colony (ABC) in support vector machine (SVM) so as to improve the performance with automatic parameter selection, where one can make full use of the advantages of ABC and SVM to further improve the accuracy rate of 3D object retrieval. Experimental results indicate that skeleton sketches can be automatically distinguished from perspective sketches, and that the proposed S3DOR-RFD method works efficiently for selected object classes.</div

MOESM3 of Tumor-infiltrating CD4+ T cells in patients with gastric cancer

Additional file 3: Figure S3. Effects of PD-1+ and Tim-3+ inhibition on IFN-Îł induction. (A) Flow cytometry results; (B) IFN-Îł induction on CD4+ cells

Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines

<div><p>Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44^high) and a main population which was either weakly positive or negative for CD44 (CD44^low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44^highCD90⁺ sub-population. Moreover, these CD44^highCD90⁺ cells revealed mesenchymal morphology, increased expression of mesenchymal markers <i>N-Cadherin</i> and <i>Vimentin</i>, increased mRNA levels of the embryonic stem cell related genes <i>Nanog</i> and <i>Oct4</i> and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44^highCD90⁺ population a good candidate for the lung CSCs. Both CD44^highCD90⁺ and CD44^highCD90⁻ cells in the PLCCL derived from SCC formed spheroids, whereas the CD44^low/− cells were lacking this potential. These results indicate that CD44^highCD90⁺ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44^high sub-population.</p> </div

Analysis of the expression of stem cell and EMT related genes in different sub-populations from the PLCCLs.

<p>Detectable expression levels of the genes were found in all sorted sub-populations from the cell lines LC004 (<b>A</b>), LC006 (<b>B</b>) and LC021 (<b>C</b>). PCR reaction without template served as a negative control. The relative expression of target genes was related to the expression of <i>PGK1</i> and normalized to the unsorted control cells. X axis shows the target genes, Y axis shows the relative expression level (RQ). The error bars reflect the variation within the triplicates, <i>P</i><0.05. (B). The FACS sorted cell sub-populations derived from the cell line LC006 were cultured in the serum-free culture system for 1–2 weeks and revealed different cell morphology for different sorted sub-populations: (a) CD44highCD90+ cells; (b) CD44highCD90− cells; (c) CD44low/−CD90+ cells, and (d) CD44low/−CD90− cells. Photomicrograph magnification, ×200.</p

Identification of CD44^high cells in the PLCCLs by flow cytometry analysis.

<p>A small sub-population of CD44^high cells could be identified in the SCLC cell line LC004 (6.63%) (<b>A</b>); the LCC cell line LC006 (2.79%) (<b>B</b>); the AC cell line LC007 (6.69%) (<b>C</b>) and the SCC cell line LC021 (14.95%) (<b>D</b>), respectively. Left panel: isotype control Ab; right panel: CD44 Ab. Data shown are from representative experiments (n>3).</p

Comparison of morphological and phenotypic features between the PLCCLs and corresponding tumor tissues.

<p><b>A.</b> H&E staining of the 4 representative primary lung cancer cell lines for different lung cancer subtypes: the SCLC cell line LC004; the LCC cell line LC006; the AC cell line LC007 and the SCC cell line LC021. The cells derived from the four subtypes of lung cancer showed heterogeneity in cellular and nuclear morphology. Cells of each primary cell line had epithelial morphology. Photomicrograph magnification, ×200. <b>B.</b> Analysis the expression of P53, Ber-EP4 and CD44 in the 4 primary cell lines and their corresponding archival patients' tumor tissues. All the primary cell lines investigated showed diffuse positive staining for P53 except the original SCC tissue of cell line LC021. Epithelial membrane antigen Ber-EP4 was widely positive in all the original lung cancer tissues and diffuse positive in all the primary cell lines. CD44 showed diffuse positive staining with different intensity in the primary cell lines and their corresponding archival patients' cancer tissues except weakly positive in the original tumor tissue of the AC cell line LC007. Sections from paraffin blocks containing human seminoma and breast cancer specimen were used as positive controls for antibodies P53, Ber-EP4 and CD44, respectively. Photomicrograph magnification, ×200.</p

Typical epithelial cells were obtained at high purity under the serum-free condition.

<p><b>A.</b> The SCLC cell line LC004; <b>B.</b> The LCC cell line LC006; <b>C.</b> The AC cell line LC007; <b>D.</b> The SCC cell line LC021. All the representative images were from primary cultured lung cancer cell lines at the second passage. Photomicrograph magnification, ×200.</p

Flow cytometry analysis of CD44 and CD90 expression in the PLCCLs.

<p>Staining by anti-CD44 FITC and anti-CD90 APC. All the cell lines showed heterogeneous staining and could be divided into 4 sub-populations: CD44^highCD90⁺, CD44^highCD90⁻, CD44^low/−CD90⁺ and CD44^low/−CD90⁻. For the SCLC cell line LC004 (<b>A</b>), the frequency of CD44^highCD90⁺ cell was 16.6%, and the CD44^highCD90⁻ cells was 8.2%. For the LCC cells line LC006 (<b>B</b>), the frequency of CD44^highCD90⁺ cells was 1.1%, the CD44^highCD90⁻ cells was 2.5%. For the AC cell line LC007 (<b>C</b>), the frequency of CD44^highCD90⁺ cells was 9.4%, and the CD44^highCD90⁻ cells was 23.4%. For the SCC cell line LC021 (<b>D</b>), the frequency of CD44^highCD90⁺ cells was 2.3%, the CD44^highCD90⁻ cells was 1.1%.</p

Relative irradiation resistance of different sorted cell populations from the cell line LC006. A.

<p>The relative resistance to irradiation of the FACS sorted CD44high and CD44low/− cells derived from the LCC cell line LC006 was compared in an X-ray radiation resistance assay. The inhibition ratios were compared among the different cell populations after irradiating the monolayer cultures at 1, 2, 3 and 4Gy. The CD44high cells displayed the higher resistance to X-ray radiation at each dose tested. <i>P</i><0.01. <b>B.</b> relative resistance to irradiation of the four different FACS sorted cell populations derived from the LCC cell line LC006 was compared in an X-ray radiation resistance assay. Four populations: CD44highCD90+, CD44highCD90−, CD44low/−CD90+ and CD44low/−CD90− cells were sorted into 96-well plates at 500 cells per well in 10 replicates. The inhibition ratios were compared among the four sub-populations after irradiation of the monolayer layer of the cultures at 1, 2 and 4Gy. The CD44highCD90+ cells displayed the highest resistance to irradiation at 2 and 4G. <i>P</i><0.01.</p