Single-cell mass cytometric analysis of peripheral immunity and multiplex plasma marker profiling of non-small cell lung cancer patients receiving PD-1 targeting immune checkpoint inhibitors in comparison with platinum-based chemotherapy.

Introduction: The effect of platinum-based chemotherapy (Chem.) and second- or multiple- line immune checkpoint PD-1 blocking therapy by Nivolumab or Pembrolizumab (ICI) was assayed in the peripheral blood of non-small cell lung cancer (NSCLC) patients. Methods: Flow cytometry was used to detect NSCLC-related antigen binding IgG antibodies. The Luminex MagPix multiplex bead-based cytokine/chemokine detecting system was used to quantitatively measure 17 soluble markers in the plasma samples. Single-cell mass cytometry was applied for the immunophenotyping of peripheral leukocytes. Results: The incubation of patient derived plasma with human NSCLC tumor cell lines, such as A549, H1975, and H1650, detected NSCLC-specific antibodies reaching a maximum of up to 32% reactive IgG-positive NSCLC cells. The following markers were detected in significantly higher concentration in the plasma of Chem. group versus healthy non-smoker and smoker controls: BTLA, CD27, CD28, CD40, CD80, CD86, GITRL, ICOS, LAG-3, PD-1, PD-L1, and TLR-2. The following markers were detected in significantly higher concentration in the plasma of ICI group versus healthy non-smoker and smoker controls: CD27, CD28, CD40, GITRL, LAG-3, PD-1, PD-L1, and TLR-2. We showed the induction of CD69 and IL-2R on CD4+ CD25+ T-cells upon chemotherapy; the exhaustion of one CD8+ T-cell population was detected by the loss of CD127 and a decrease in CD27. CD19+CD20+, CD79B+, or activated B-cell subtypes showed CD69 increase and downregulation of BTLA, CD27, and IL-2R in NSCLC patients following chemotherapy or ICI. Discussion: Peripheral immunophenotype caused by chemotherapy or PD-1 blocking was shown in the context of advanced NSCLC.

Comparison of humoral and cellular immune responses in hematologic diseases following completed vaccination protocol with BBIBP-CorV, or AZD1222, or BNT162b2 vaccines against SARS-CoV-2.

Background: Vaccination has proven the potential to control the COVID-19 pandemic worldwide. Although recent evidence suggests a poor humoral response against SARS-CoV-2 in vaccinated hematological disease (HD) patients, data on vaccination in these patients is limited with the comparison of mRNA-based, vector-based or inactivated virus-based vaccines. Methods: Forty-nine HD patients and 46 healthy controls (HCs) were enrolled who received two-doses complete vaccination with BNT162b2, or AZD1222, or BBIBP-CorV, respectively. The antibodies reactive to the receptor binding domain of spike protein of SARS-CoV-2 were assayed by Siemens ADVIA Centaur assay. The reactive cellular immunity was assayed by flow cytometry. The PBMCs were reactivated with SARS-CoV-2 antigens and the production of activation-induced markers (TNF-α, IFN-γ, CD40L) was measured in CD4+ or CD8+ T-cells ex vivo. Results: The anti-RBD IgG level was the highest upon BNT162b2 vaccination in HDs (1264 BAU/mL) vs. HCs (1325 BAU/mL) among the studied groups. The BBIBP-CorV vaccination in HDs (339.8 BAU/mL ***p < 0.001) and AZD1222 in HDs (669.9 BAU/mL *p < 0.05) resulted in weaker antibody response vs. BNT162b2 in HCs. The response rate of IgG production of HC vs. HD patients above the diagnostic cut-off value was 100% vs. 72% for the mRNA-based BNT162b2 vaccine; 93% vs. 56% for the vector-based AZD1222, or 69% vs. 33% for the inactivated vaccine BBIBP-CorV, respectively. Cases that underwent the anti-CD20 therapy resulted in significantly weaker (**p < 0.01) anti-RBD IgG level (302 BAU/mL) than without CD20 blocking in the HD group (928 BAU/mL). The response rates of CD4+ TNF-α+, CD4+ IFN-γ+, or CD4+ CD40L+ cases were lower in HDs vs. HCs in all vaccine groups. However, the BBIBP-CorV vaccine resulted the highest CD4+ TNF-α and CD4+ IFN-γ+ T-cell mediated immunity in the HD group. Conclusion: We have demonstrated a significant weaker overall response to vaccines in the immunologically impaired HD population vs. HCs regardless of vaccine type. Although, the humoral immune activity against SARS-CoV-2 can be highly evoked by mRNA-based BNT162b2 vaccination compared to vector-based AZD1222 vaccine, or inactivated virus vaccine BBIBP-CorV, whereas the CD4+ T-cell mediated cellular activity was highest in HDs vaccinated with BBIBP-CorV.

Introduction of an Ultraviolet C-Irradiated 4T1 Murine Breast Cancer Whole-Cell Vaccine Model.

The advent of immunotherapy has revolutionized cancer treatments. However, the application of immune checkpoint inhibitors may entail severe side effects, with the risk of therapeutic resistance. The generation of chimeric antigen receptor (CAR) T-cells or CAR-NK cells requires specialized molecular laboratories, is costly, and is difficult to adapt to the rapidly growing number of cancer patients. To provide a simpler but effective immune therapy, a whole-cell tumor vaccine protocol was established based on ultraviolet C (UCV)-irradiated 4T1 triple-negative breast cancer cells. The apoptosis of tumor cells after UVC irradiation was verified using resazurin and Annexin V/propidium iodide flow cytometric assays. Protective immunity was achieved in immunized BALB/c mice, showing partial remission. Adoptive transfer of splenocytes or plasma from the mice in remission showed a protective effect in the naive BALB/c mice that received a living 4T1 tumor cell injection. 4T1-specific IgG antibodies were recorded in the plasma of the mice following immunization with the whole-cell vaccine. Interleukin-2 (IL-2) and oligonucleotide 2006 (ODN2006) adjuvants were used for the transfer of splenocytes from C57BL/6 mice into cyclophosphamide-treated BALB/c mice, resulting in prolonged survival, reduced tumor growth, and remission in 33% of the cases, without the development of the graft-versus-host disease. Our approach offers a simple, cost-effective whole-cell vaccine protocol that can be administered to immunocompetent healthy organisms. The plasma or the adoptive transfer of HLA-matching immunized donor-derived leukocytes could be used as an immune cell therapy for cancer patients.

Single-cell immunophenotyping revealed the association of CD4+ central and CD4+ effector memory T cells linking exacerbating chronic obstructive pulmonary disease and NSCLC.

Introduction: Tobacco smoking generates airway inflammation in chronic obstructive pulmonary disease (COPD), and its involvement in the development of lung cancer is still among the leading causes of early death. Therefore, we aimed to have a better understanding of the disbalance in immunoregulation in chronic inflammatory conditions in smoker subjects with stable COPD (stCOPD), exacerbating COPD (exCOPD), or non-small cell lung cancer (NSCLC). Methods: Smoker controls without chronic illness were recruited as controls. Through extensive mapping of single cells, surface receptor quantification was achieved by single-cell mass cytometry (CyTOF) with 29 antibodies. The CyTOF characterized 14 main immune subsets such as CD4+, CD8+, CD4+/CD8+, CD4-/CD8-, and γ/δ T cells and other subsets such as CD4+ or CD8+ NKT cells, NK cells, B cells, plasmablasts, monocytes, CD11cdim, mDCs, and pDCs. The CD4+ central memory (CM) T cells (CD4+/CD45RA-/CD45RO+/CD197+) and CD4+ effector memory (EM) T cells (CD4+/CD45RA-/CD45RO+/CD197-) were FACS-sorted for RNA-Seq analysis. Plasma samples were assayed by Luminex MAGPIX® for the quantitative measurement of 17 soluble immuno-oncology mediators (BTLA, CD28, CD80, CD27, CD40, CD86, CTLA-4, GITR, GITRL, HVEM, ICOS, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, TLR-2) in the four studied groups. Results: Our focus was on T-cell-dependent differences in COPD and NSCLC, where peripheral CD4+ central memory and CD4+ effector memory cells showed a significant reduction in exCOPD and CD4+ CM showed elevation in NSCLC. The transcriptome analysis delineated a perfect correlation of differentially expressed genes between exacerbating COPD and NSCLC-derived peripheral CD4+ CM or CD4+ EM cells. The measurement of 17 immuno-oncology soluble mediators revealed a disease-associated phenotype in the peripheral blood of stCOPD, exCOPD, and NSCLC patients. Discussion: The applied single-cell mass cytometry, the whole transcriptome profiling of peripheral CD4+ memory cells, and the quantification of 17 plasma mediators provided complex data that may contribute to the understanding of the disbalance in immune homeostasis generated or sustained by tobacco smoking in COPD and NSCLC.

A cytotoxic survey on 2-amino-1H-imidazol based synthetic marine sponge alkaloid analogues.

Here, we describe the synthesis and biologic activity evaluation of 20 novel synthetic marine sponge alkaloid analogues with 2-amino-1H-imidazol (2-AI) core. Cytotoxicity was tested on murine 4T1 breast cancer, A549 human lung cancer, and HL-60 human myeloid leukemia cells by the resazurin assay. A total of 18 of 20 compounds showed cytotoxic effect on the cancer cell lines with different potential. Viability of healthy human fibroblasts and peripheral blood mononuclear cells upon treatment was less hampered compared to cancer cell lines supporting tumor cell specific cytotoxicity of our compounds. The most cytotoxic compounds resulted the following IC50 values 28: 2.91 µM on HL-60 cells, and 29: 3.1 µM on 4T1 cells. The A549 cells were less sensitive to the treatments with IC50 15 µM for both 28 and 29. Flow cytometry demonstrated the apoptotic effect of the most active seven compounds inducing phosphatidylserine exposure and sub-G1 fragmentation of nuclear DNA. Cell cycle arrest was also observed. Four compounds caused depolarization of the mitochondrial membrane potential as an early event of apoptosis. Two lead compounds inhibited tumor growth in vivo in the 4T1 triple negative breast cancer and A549 human lung adenocarcinoma xenograft models. Novel marine sponge alkaloid analogues are demonstrated as potential anticancer agents for further development.

Comparison of Homologous and Heterologous Booster SARS-CoV-2 Vaccination in Autoimmune Rheumatic and Musculoskeletal Patients.

Vaccination against SARS-CoV-2 to prevent COVID-19 is highly recommended for immunocompromised patients with autoimmune rheumatic and musculoskeletal diseases (aiRMDs). Little is known about the effect of booster vaccination or infection followed by previously completed two-dose vaccination in aiRMDs. We determined neutralizing anti-SARS-CoV-2 antibody levels and applied flow cytometric immunophenotyping to quantify the SARS-CoV-2 reactive B- and T-cell mediated immunity in aiRMDs receiving homologous or heterologous boosters or acquired infection following vaccination. Patients receiving a heterologous booster had a higher proportion of IgM+ SARS-CoV-2 S+ CD19+CD27+ peripheral memory B-cells in comparison to those who acquired infection. Biologic therapy decreased the number of S+CD19+; S+CD19+CD27+IgG+; and S+CD19+CD27+IgM+ B-cells. The response rate to a booster event in cellular immunity was the highest in the S-, M-, and N-reactive CD4+CD40L+ T-cell subset. Patients with a disease duration of more than 10 years had higher proportions of CD8+TNF-α+ and CD8+IFN-γ+ T-cells in comparison to patients who were diagnosed less than 10 years ago. We detected neutralizing antibodies, S+ reactive peripheral memory B-cells, and five S-, M-, and N-reactive T-cells subsets in our patient cohort showing the importance of booster events. Biologic therapy and <10 years disease duration may confound anti-SARS-CoV-2 specific immunity in aiRMDs.

Humoral and Cellular Immunogenicity and Safety of Five Different SARS-CoV-2 Vaccines in Patients With Autoimmune Rheumatic and Musculoskeletal Diseases in Remission or With Low Disease Activity and in Healthy Controls: A Single Center Study.

Background: Vaccine-induced immunity is essential for controlling the COVID-19 pandemic. Data on humoral and cellular immunogenicity and safety of different SARS-CoV-2 vaccines in patients with autoimmune rheumatic and musculoskeletal diseases (RMDs) are limited. Methods: A single center observational study evaluated the immunogenicity and safety of the two-dose regimen of the BBIBP-CorV inactivated, Gam-COVID-Vac and AZD1222 adenovirus-based, and BNT162b2 and mRNA-1273 mRNA-based vaccines in patients with RMDs (n = 89) compared with healthy controls (n = 74). Neutralizing anti-RBD (receptor binding domain) specific antibodies and SARS-CoV-2 specific T-cell response were measured one and four months after the second vaccine dose in parallel with vaccination efficacy and safety. Results: Disease-specific comparison showed that antibody response at four months was higher in spondylarthropathies compared to rheumatoid arthritis and autoimmune RMDs. Risk factors for reduced immunogenicity included longer disease duration, positive immunoserological profile and anti-CD20 therapy of patients. The rate of positive anti-RBD antibody response for healthy controls versus patients after 4 months post vaccination was 69% vs. 55% for the inactivated viral vaccine BBIBP-CorV, 97% vs. 53% for the pooled data of adenovirus vector-based vaccines Gam-COVID-Vac and AZD1222, or 100% vs. 81% for the pooled data of mRNA vaccines BNT162b2 and mRNA-1273, respectively. Patients who received the Gam-COVID-Vac or mRNA-1273 vaccines had a higher proportion of TNF-α producing CD4+ T-cells upon SARS-CoV-2 antigen stimulation compared to the inactivated viral vaccine. Conclusion: All five investigated vaccines were immunogenic in the majority of patients and healthy controls with variable antibody and T-cell response and an acceptable safety profile.

Chronic Obstructive Pulmonary Disease: Epidemiology, Biomarkers, and Paving the Way to Lung Cancer.

Chronic obstructive pulmonary disease (COPD), the frequently fatal pathology of the respiratory tract, accounts for half a billion cases globally. COPD manifests via chronic inflammatory response to irritants, frequently to tobacco smoke. The progression of COPD from early onset to advanced disease leads to the loss of the alveolar wall, pulmonary hypertension, and fibrosis of the respiratory epithelium. Here, we focus on the epidemiology, progression, and biomarkers of COPD with a particular connection to lung cancer. Dissecting the cellular and molecular players in the progression of the disease, we aim to shed light on the role of smoking, which is responsible for the disease, or at least for the more severe symptoms and worse patient outcomes. We summarize the inflammatory conditions, as well as the role of EMT and fibroblasts in establishing a cancer-prone microenvironment, i.e., the soil for 'COPD-derived' lung cancer. We highlight that the major health problem of COPD can be alleviated via smoking cessation, early diagnosis, and abandonment of the usage of biomass fuels on a global basis.

[Single cell mass cytometric comparison of human H1975 lung and MDA-MD-231 breast adenocarcinoma cellular models]. / A humán H1975 tüdo- és MDA-MB-231 emloadenokarcinóma-sejtes modellek egysejt-tömegcitometriás összehasonlító elemzése.

The most frequent cancer types are lung and breast cancer in the Western world. However, the prognosis of breast cancer patients shows an improved tendency, while lung cancer types remained with high mortality. Intratumor heterogeneity (ITH) frequently leads to the failure of treatments, so there is an unmet need revealing ITH at single cell resolution. Our aim was to study female-derived human H1975 lung and MDA-MB-231 triple-negative breast cancer adenocarcinoma cell line models using single cell mass cytometry. Nine of thirteen carcinoma markers showed significant differences in the percentage of cells. Our current work shed light on the intra- and inter cell line heterogeneity still preserved in the studied, widely-used adenocarcinoma laboratory models.

Antiproliferative and antimetastatic characterization of an exo-heterocyclic androstane derivative against human breast cancer cell lines.

Cancer in general, and specifically gynaecological neoplasms, represents a major public health issue worldwide. Based on the effect of sex hormones on breast tumorigenesis and prognosis, as well as on the development of breast cancer metastases, modification of the steroid skeleton is a hotspot of research for novel anticancer agents. Numerous recent studies support that minor modifications of the androstane skeleton yield potent antiproliferative and antimetastatic drug candidates. The aim of the present study was to assess the antitumor and antimetastatic properties, as well as the mechanism of action of a D-ring-modified exo-heterocyclic androstadiene derivative named 17APAD. The test compound was found to be highly selective towards human breast cancer-derived cell lines (MCF-7, T47D, MDA-MB-361, MDA-MB-231) compared to non-cancerous fibroblast cells (NIH/3T3), and exerted superior effect compared to the clinically applied reference drug cisplatin. Changes in MCF-7- and MDA-MB-231 cell morphology and membrane integrity induced by the test substance were assessed by fluorescent double staining. Cell cycle disturbances were analyzed by flow cytometry, and concentration-dependent alterations were detected on breast cancer cell lines. Mitochondrial apoptosis induced by the test compound was demonstrated by JC-1 staining. Inhibitory effects on metastasis formation, including the inhibition of migration, invasion and intravasation were investigated in 2D and 3D models. Significant anti-migratory and anti-invasive effects on MCF-7 and MDA-MB-231 cells were detected after 24 h exposure in 2D wound healing and Boyden-chamber assays. The anti-intravasative properties of 17APAD were evident after 4 h of incubation in a co-culture 3D circular chemorepellent-induced defects (CCID) assay, and the level of inhibition at concentrations ≥2 µM was comparable to that exerted by the focal adhesion kinase inhibitor defactinib. Single cell mass cytometry revealed that chemosensitive subpopulations of MDA-MB-231 cells engaged to apoptosis were less positive for EGFR, CD274, and CD326, while the percentage of cells positive for GLUT1, MCT4, Pan-Keratin, CD66(a,c,e), Galectin-3 and TMEM45A increased in response to 17APAD treatment. Finally, the novel androstane analogue 17APAD had an outstanding inhibitory effect on tumour growth in the 4T1 orthotopic murine breast cancer model in vivo after 2 weeks of intraperitoneal administration. These findings support that substitution of the androsta-5,16-diene framework with a N-containing heterocyclic moiety at C17 position yields a molecular entity rational to be considered for design and synthesis of novel, effective antitumor agents, and 17APAD is worth further investigation as a promising anticancer drug candidate.

Analysis of the Single-Cell Heterogeneity of Adenocarcinoma Cell Lines and the Investigation of Intratumor Heterogeneity Reveals the Expression of Transmembrane Protein 45A (TMEM45A) in Lung Adenocarcinoma Cancer Patients.

Intratumoral heterogeneity (ITH) is responsible for the majority of difficulties encountered in the treatment of lung-cancer patients. Therefore, the heterogeneity of NSCLC cell lines and primary lung adenocarcinoma was investigated by single-cell mass cytometry (CyTOF). First, we studied the single-cell heterogeneity of frequent NSCLC adenocarcinoma models, such as A549, H1975, and H1650. The intra- and inter-cell-line single-cell heterogeneity is represented in the expression patterns of 13 markers-namely GLUT1, MCT4, CA9, TMEM45A, CD66, CD274 (PD-L1), CD24, CD326 (EpCAM), pan-keratin, TRA-1-60, galectin-3, galectin-1, and EGFR. The qRT-PCR and CyTOF analyses revealed that a hypoxic microenvironment and altered metabolism may influence cell-line heterogeneity. Additionally, human primary lung adenocarcinoma and non-involved healthy lung tissue biopsies were homogenized to prepare a single-cell suspension for CyTOF analysis. The CyTOF showed the ITH of human primary lung adenocarcinoma for 14 markers; particularly, the higher expressions of GLUT1, MCT4, CA9, TMEM45A, and CD66 were associated with the lung-tumor tissue. Our single-cell results are the first to demonstrate TMEM45A expression in human lung adenocarcinoma, which was verified by immunohistochemistry.

Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model.

The treatment of metastatic breast cancer remained a challenge despite the recent breakthrough in the immunotherapy regimens. Here, we addressed the multidimensional immunophenotyping of 4T1 metastatic breast cancer by the state-of-the-art single cell mass cytometry (CyTOF). We determined the dose and time dependent cytotoxicity of cisplatin on 4T1 cells by the xCelligence real-time electronic sensing assay. Cisplatin treatment reduced tumor growth, number of lung metastasis, and the splenomegaly of 4T1 tumor bearing mice. We showed that cisplatin inhibited the tumor stroma formation, the polarization of carcinoma-associated fibroblasts by the diminished proteolytic activity of fibroblast activating protein. The CyTOF analysis revealed the emergence of CD11b+/Gr-1+/CD44+ or CD11b+/Gr-1+/IL-17A+ myeloid-derived suppressor cells (MDSCs) and the absence of B220+ or CD62L+ B-cells, the CD62L+/CD4+ and CD62L+/CD8+ T-cells in the spleen of advanced cancer. We could show the immunomodulatory effect of cisplatin via the suppression of splenic MDSCs and via the promotion of peripheral IFN-γ+ myeloid cells. Our data could support the use of low dose chemotherapy with cisplatin as an immunomodulatory agent for metastatic triple negative breast cancer.

Single Cell Mass Cytometry of Non-Small Cell Lung Cancer Cells Reveals Complexity of In vivo And Three-Dimensional Models over the Petri-dish.

Single cell genomics and proteomics with the combination of innovative three-dimensional (3D) cell culture techniques can open new avenues toward the understanding of intra-tumor heterogeneity. Here, we characterize lung cancer markers using single cell mass cytometry to compare different in vitro cell culturing methods: two-dimensional (2D), carrier-free, or bead-based 3D culturing with in vivo xenografts. Proliferation, viability, and cell cycle phase distribution has been investigated. Gene expression analysis enabled the selection of markers that were overexpressed: TMEM45A, SLC16A3, CD66, SLC2A1, CA9, CD24, or repressed: EGFR either in vivo or in long-term 3D cultures. Additionally, TRA-1-60, pan-keratins, CD326, Galectin-3, and CD274, markers with known clinical significance have been investigated at single cell resolution. The described twelve markers convincingly highlighted a unique pattern reflecting intra-tumor heterogeneity of 3D samples and in vivo A549 lung cancer cells. In 3D systems CA9, CD24, and EGFR showed higher expression than in vivo. Multidimensional single cell proteome profiling revealed that 3D cultures represent a transition from 2D to in vivo conditions by intermediate marker expression of TRA-1-60, TMEM45A, pan-keratin, CD326, MCT4, Gal-3, CD66, GLUT1, and CD274. Therefore, 3D cultures of NSCLC cells bearing more putative cancer targets should be used in drug screening as the preferred technique rather than the Petri-dish.