scholarly journals Exosomal Hsp70 in Liquid Biopsies - A Biomarker for Prediction and Response Monitoring in Cancer

2021 ◽  
Author(s):  
Caroline Werner ◽  
Stefan Stangl ◽  
Lukas Salvermoser ◽  
Melissa Schwab ◽  
Maxim Shevstov ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Conformational Changes ◽  
Small Cell Lung Carcinoma ◽  
Sandwich Elisa ◽  
Response Monitoring ◽  
Tumor Biomarker ◽  
Liquid Biopsies ◽  
Cell Lung Carcinoma ◽  
Viable Tumor ◽  
Lipid Environment

In contrast to normal cells, tumor cells of multiple entities overexpress the Heat Shock Protein 70 (Hsp70) not only in the cytosol, but also present it on their plasma membrane in a tumor-specific manner. Furthermore, membrane-Hsp70 positive tumor cells actively release Hsp70 into lipid microvesicles termed exosomes into the blood. Due to conformational changes of Hsp70 in the lipid environment, most commercially available antibodies fail to detect membrane-bound and exosomal Hsp70. To fill this gap and to assess the role of exosomal Hsp70 in the circulation as a potential tumor biomarker, we established the novel complete Hsp70 (compHsp70) sandwich ELISA using two monoclonal antibodies (mAbs) that are able to recognize both, free and lipid-associated Hsp70 on the cell surface of viable tumor cells and exosomes. The epitopes of the mAbs cmHsp70.1 (aa 451-461) and cmHsp70.2 (aa 614-623) that are conserved among different species reside in the substrate-binding domain of Hsp70, with measured affinities of 0.42 nM and 0.44 nM, respectively. Validation of the compHsp70 ELISA revealed a high intra- and inter-assay precision, linearity in a concentration range of 1.56 to 25 ng/ml, high recovery rates of ‘spiked’ liposomal Hsp70 (>84%), comparable values between human serum and plasma samples, and no interference by food intake or age of the donors. Hsp70 concentrations in the circulation of patients with glioblastoma, squamous cell or adeno non-small cell lung carcinoma (NSCLC) at diagnosis were significantly higher than those of healthy volunteers. Hsp70 concentrations dropped concomitantly with the decrease in viable tumor mass on irradiation of patients with approximately 20 Gy (range 18 – 22.5 Gy) or after completion of radiotherapy (60 - 70 Gy). In summary, the compHsp70 ELISA presented herein provides a highly sensitive and reliable tool for measuring free and exosomal Hsp70 in liquid biopsies of tumor patients, levels of which can be used as a predictive tumor-specific biomarker, risk assessment and for monitoring therapeutic outcome.

scholarly journals Hsp70 in Liquid Biopsies—A Tumor-Specific Biomarker for Detection and Response Monitoring in Cancer

Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3706
Author(s):  
Caroline Werner ◽  
Stefan Stangl ◽  
Lukas Salvermoser ◽  
Melissa Schwab ◽  
Maxim Shevtsov ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Extracellular Vesicles ◽  
Conformational Changes ◽  
Small Cell Lung Carcinoma ◽  
Sandwich Elisa ◽  
Response Monitoring ◽  
Tumor Biomarker ◽  
Liquid Biopsies ◽  
Cell Lung Carcinoma ◽  
Viable Tumor

In contrast to normal cells, tumor cells of multiple entities overexpress the Heat shock protein 70 (Hsp70) not only in the cytosol, but also present it on their plasma membrane in a tumor-specific manner. Furthermore, membrane Hsp70-positive tumor cells actively release Hsp70 in small extracellular vesicles with biophysical characteristics of exosomes. Due to conformational changes of Hsp70 in a lipid environment, most commercially available antibodies fail to detect membrane-bound and vesicular Hsp70. To fill this gap and to assess the role of vesicular Hsp70 in circulation as a potential tumor biomarker, we established the novel complete (comp)Hsp70 sandwich ELISA, using two monoclonal antibodies (mAbs), that is able to recognize both free and lipid-associated Hsp70 on the cell surface of viable tumor cells and on small extracellular vesicles. The epitopes of the mAbs cmHsp70.1 (aa 451–461) and cmHsp70.2 (aa 614–623) that are conserved among different species reside in the substrate-binding domain of Hsp70 with measured affinities of 0.42 nM and 0.44 nM, respectively. Validation of the compHsp70 ELISA revealed a high intra- and inter-assay precision, linearity in a concentration range of 1.56 to 25 ng/mL, high recovery rates of spiked liposomal Hsp70 (>84%), comparable values between human serum and plasma samples and no interference by food intake or age of the donors. Hsp70 concentrations in the circulation of patients with glioblastoma, squamous cell or adeno non-small cell lung carcinoma (NSCLC) at diagnosis were significantly higher than those of healthy donors. Hsp70 concentrations dropped concomitantly with a decrease in viable tumor mass upon irradiation of patients with approximately 20 Gy (range 18–22.5 Gy) and after completion of radiotherapy (60–70 Gy). In summary, the compHsp70 ELISA presented herein provides a sensitive and reliable tool for measuring free and vesicular Hsp70 in liquid biopsies of tumor patients, levels of which can be used as a tumor-specific biomarker, for risk assessment (i.e., differentiation of grade III vs. IV adeno NSCLC) and monitoring of therapeutic outcomes.

scholarly journals Cytokeratin-positive interstitial reticulum cell (CIRC) tumor in the lymph node: a case report of the transformation from the epithelioid cell type to the spindle cell type

2020 ◽  
Vol 15 (1) ◽  
Author(s):  
Sachiko Kaji ◽  
Nobuyuki Hiruta ◽  
Daisuke Sasai ◽  
Makoto Nagashima ◽  
Rintaro Ohe ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Lung Carcinoma ◽  
Spindle Cell ◽  
Small Cell Lung Carcinoma ◽  
Epithelioid Cell ◽  
Small Cell ◽  
Cell Type ◽  
Small Cell Lung ◽  
Cell Lung Carcinoma ◽  
Spindle Cell Type

Abstract Background Cytokeratin-positive interstitial reticulum cells (CIRCs), which are a subgroup of fibroblastic reticular cells (FRCs), are known to be present in the lymph nodes. There have been only a few cases of tumors derived from CIRCs. Case presentation We have reported a new case involving a CIRC tumor in a 75-year-old man and reviewed the literature. The resected mediastinal lymph nodes showed epithelial-like proliferation of large atypical round and polygonal epithelioid cells. The tumor cells expressed CK8, CK18, CAM5.2, AE1/AE3, epithelial membrane antigen, vimentin, fascin, and some FRC markers, which is consistent with the diagnosis of a CIRC tumor. Following chemotherapy, the CIRC tumor was observed to have responded very well and became difficult to confirm on imaging, but a small cell lung carcinoma developed 12 months later. Chemoradiotherapy was performed, but the patient passed away 29 months after the initial diagnosis. The autopsy revealed the recurrence of the CIRC tumor, residual small cell lung carcinoma, and a very small latent carcinoma of the prostate. The relapsed CIRC tumor cells had a spindle shape; they were highly pleomorphic and had invaded the superior vena cava. Conclusion We first reported autopsy findings of CIRC tumors and demonstrated the transformation of the tumor from the epithelioid cell type to the spindle cell type.

scholarly journals Induction of CD8 T-cell-Ifn-γ response and positive clinical outcome after immunization with gene-modified allogeneic tumor cells in advanced non-small-cell lung carcinoma

2003 ◽  
Vol 10 (11) ◽  
pp. 850-858 ◽  
Author(s):  
Luis E Raez ◽  
Peter A Cassileth ◽  
James J Schlesselman ◽  
Swaminathan Padmanabhan ◽  
Eva Z Fisher ◽  
...  
Keyword(s):  
T Cell ◽  
Clinical Outcome ◽  
Tumor Cells ◽  
Lung Carcinoma ◽  
Small Cell Lung Carcinoma ◽  
Cd8 T Cell ◽  
Small Cell ◽  
Small Cell Lung ◽  
Cell Lung Carcinoma ◽  
Ifn Γ

scholarly journals Antigen-dependent CD28 Signaling Selectively Enhances Survival and Proliferation in Genetically Modified Activated Human Primary T Lymphocytes

1998 ◽  
Vol 188 (4) ◽  
pp. 619-626 ◽  
Author(s):  
Anja Krause ◽  
Hong-Fen Guo ◽  
Jean-Baptiste Latouche ◽  
Cuiwen Tan ◽  
Nai-Kong  V. Cheung ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Tumor Cells ◽  
Small Cell Lung Carcinoma ◽  
Interleukin 2 ◽  
Culture Conditions ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Cell Lung Carcinoma ◽  
Antiidiotypic Antibody ◽  
Antigen Presenting

Most tumor cells function poorly as antigen-presenting cells in part because they do not express costimulatory molecules. To provide costimulation to T lymphocytes that recognize tumor cells, we constructed a CD28-like receptor specific for GD2, a ganglioside overexpressed on the surface of neuroblastoma, small-cell lung carcinoma, melanoma, and other human tumors. Recognition of GD2 was provided by a single-chain antibody derived from the GD2-specific monoclonal antibody 3G6. We demonstrate that the chimeric receptor 3G6-CD28 provides CD28 signaling upon specific recognition of the GD2 antigen on tumor cells. Human primary T lymphocytes retrovirally transduced with 3G6-CD28 secrete interleukin 2, survive proapoptotic culture conditions, and selectively undergo clonal expansion in the presence of an antiidiotypic antibody specific for 3G6-CD28. Polyclonal CD8+ lymphocytes expressing 3G6-CD28 are selectively expanded when cultured with cells expressing allogeneic major histocompatibility complex class I together with GD2. Primary T cells given such an antigen-dependent survival advantage should be very useful to augment immune responses against tumor cells.

scholarly journals Early detection of the major male cancer types in blood-based liquid biopsies using a DNA methylation panel

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Vera Constâncio ◽  
Sandra P. Nunes ◽  
Catarina Moreira-Barbosa ◽  
Rui Freitas ◽  
Jorge Oliveira ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Early Detection ◽  
Minimally Invasive ◽  
Lung Carcinoma ◽  
Small Cell Lung Carcinoma ◽  
Small Cell ◽  
Small Cell Lung ◽  
Liquid Biopsies ◽  
Cell Lung Carcinoma ◽  
Cancer Types

Abstract Background Lung (LC), prostate (PCa) and colorectal (CRC) cancers are the most incident in males worldwide. Despite recent advances, optimal population-based cancer screening methods remain an unmet need. Due to its early onset, cancer specificity and accessibility in body fluids, aberrant DNA promoter methylation might be a valuable minimally invasive tool for early cancer detection. Herein, we aimed to develop a minimally invasive methylation-based test for simultaneous early detection of LC, PCa and CRC in males, using liquid biopsies. Results Circulating cell-free DNA was extracted from 102 LC, 121 PCa and 100 CRC patients and 136 asymptomatic donors’ plasma samples. Sodium-bisulfite modification and whole-genome amplification was performed. Promoter methylation levels of APCme, FOXA1me, GSTP1me, HOXD3me, RARβ2me, RASSF1Ame, SEPT9me and SOX17me were assessed by multiplex quantitative methylation-specific PCR. SEPT9me and SOX17me were the only biomarkers shared by all three cancer types, although they detected CRC with limited sensitivity. A “PanCancer” panel (FOXA1me, RARβ2me and RASSF1Ame) detected LC and PCa with 64% sensitivity and 70% specificity, complemented with “CancerType” panel (GSTP1me and SOX17me) which discriminated between LC and PCa with 93% specificity, but with modest sensitivity. Moreover, a HOXD3me and RASSF1Ame panel discriminated small cell lung carcinoma from non-small cell lung carcinoma with 75% sensitivity, 88% specificity, 6.5 LR+ and 0.28 LR–. An APCme and RASSF1Ame panel independently predicted disease-specific mortality in LC patients. Conclusions We concluded that a DNA methylation-based test in liquid biopsies might enable minimally invasive screening of LC and PCa, improving patient compliance and reducing healthcare costs. Moreover, it might assist in LC subtyping and prognostication.

Imaging of the P-glycoprotein in normal and multidrug-resistant small-cell lung carcinoma cells with the Atomic-Force Microscope(AFM)

1994 ◽  
Vol 52 ◽  
pp. 1074-1075
Author(s):  
J. M. Tsai ◽  
D. Sadava ◽  
S. A. C. Gould
Keyword(s):  
Tumor Cells ◽  
Small Cell Lung Carcinoma ◽  
Protein A ◽  
Multidrug Resistant ◽  
Extracellular Medium ◽  
Contact Mode ◽  
Cell Lung Carcinoma ◽  
Lung Carcinoma Cells ◽  
Atomic Force ◽  
P Glycoprotein

Cancer is characterized by the often rapid and uncontrolled rate of cell growth. This alteration in growth pattern causes normal cells to become tumor cells. After undergoing a period of chemotherapy, some tumor cells become resistant to a variety of drugs, a phenomenon known as the multidrug resistance (MDR). One explanation for this change is the overexpression of P-glycoprotein in the drug-resistant cells. This membrane protein is capable of pumping drugs into the extracellular medium. Since the drugs do not accumulate, the tumor cells are not killed. In order to examine this protein, a contact mode AFM was used to image the cell membranes of both the normal and the MDR tumor cells. The four figures provided show the topographical information of the membranes and suggest that there are differences between the cell lines.

scholarly journals Combination of tissue and liquid biopsy molecular profiling to detect transformation to small cell lung carcinoma during osimertinib treatment

2020 ◽  
Vol 12 ◽  
pp. 175883592097419
Author(s):  
Julie A. Vendrell ◽  
Xavier Quantin ◽  
Isabelle Serre ◽  
Jérôme Solassol
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
Liquid Biopsy ◽  
Small Cell Lung Carcinoma ◽  
Small Cell ◽  
Small Cell Lung ◽  
Liquid Biopsies ◽  
Cell Lung Carcinoma ◽  
Histological Transformation

Background: Histological transformation of advanced non-small cell lung cancer (NSCLC) to small cell lung cancer (SCLC) is one of the mechanisms of resistance to third-generation tyrosine kinase inhibitors (TKIs), such as osimertinib. This acquired TKI resistance is linked to the high degree of tumor heterogeneity and adaptive cellular signaling pathways, including epidermal growth factor receptor ( EGFR)-dependent pathways, observed in NSCLC. Methods: Here, we investigated a series of paired pre- and post-histological transformation biopsies obtained from three patients initially having a NSCLC with an EGFRactivating mutation treated with first-generation TKI, who then received osimertinib as second-line after EGFRT790M resistance and, lastly, developed a histological transformation to SCLC. Both tissue and liquid biopsies were analyzed using large panel sequencing approaches at various time points to reconstruct the clonal evolutionary history of the tumor. Results: Our complementary analysis of tumor tissue and circulating tumor DNA samples allowed us to better characterize the histological and molecular alterations associated with resistance to osimertinib. SCLC transformation was linked to the presence of several concomitant gene alterations, including EGFR, TP53 and RB1, but also to specific signal bypass, such as EGFR and MET amplifications and activation of the PI3K/AKT/mTOR pathway. Conclusion: Our report emphasizes the mutational landscape of SCLC histological transformation and highlights the importance of combining tissue and liquid biopsy profiling before and during osimertinib treatment to predict such histological transformation.

scholarly journals Concordance of PD-L1 expression and CD8+ TIL intensity between NSCLC and synchronous brain metastases

2019 ◽  
Author(s):  
Sebnem Batur ◽  
Onur Dulger ◽  
Sermin Durak ◽  
Perran Fulden Yumuk ◽  
Hale Basak Caglar ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Lung Tumor ◽  
Small Cell Lung Carcinoma ◽  
Tumor Infiltrating Lymphocytes ◽  
Predictive Biomarker ◽  
Lung Tumors ◽  
Strongly Correlated ◽  
Cell Lung Carcinoma ◽  
Cutoff Values ◽  
Primary Lung Tumors

Programmed death-ligand 1 (PD-L1) is suggested to be a predictive biomarker in non-small-cell lung carcinoma (NSCLC). However, the differential expression of PD-L1 in primary lung tumor vs. synchronous metastases, especially brain metastasis (BM), remains unclear. This study assessed the concordance of PD-L1 expression on tumor cells and tumor infiltrating lymphocytes (TILs) and CD8+ TIL intensity between primary lung tumors and synchronous BMs from 24 NSCLC patients. PD-L1, CD3, and CD8 positivity was determined by immunohistochemistry (IHC). PD-L1 scoring was based on the proportion of tumor cells with membranous expression of PD-L1 and the cutoff values <1%, 1–49%, and ≥50%. CD3 and CD8 positivity in TILs was evaluated semi-quantitatively and the proportion of CD3+/CD8+ TILs was determined. PD-L1 expression on tumor cells and TILs was evaluated in relation to CD3+/CD8+ TIL proportions and the intensity of CD8+ TILs between the paired primary lung and BM tissues. In the primary lung tumors, PD-L1 positivity was observed in 25%, 37.5%, and 37.5% cases for the cutoff values <1%, 1–49%, and ≥50%, respectively. PD-L1 expression on tumor cells was strongly correlated between the paired primary lung and BM tissues, in all cutoff groups. However, PD-L1 expression on TILs and the proportion of CD3+/CD8+ TILs were not strongly correlated in all three groups between the paired primary lung tumors and BMs. The intensity of CD8+ TILs was concordant in only 54.16% of the paired primary lung tumors and BMs. This study showed a high concordance of PD-L1 expression in neoplastic cells between primary NSCLC and synchronous BMs.

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