Development of Ex Vivo Analysis for Examining Cell Composition, Immunological Landscape, Tumor and Immune Related Markers in Non-Small-Cell Lung Cancer
<p>Representative images demonstrate isolation of cancer cells (<b>a</b>) from surgically resected tumor sample sq1 (weight 0.07 g), which is indicated by the red arrow in the Petri dish 5 cm in diameter, (<b>b</b>) in the cell suspension after separating fibrotic tissue in a sieve and (<b>c</b>–<b>f</b>) their analysis on (<b>c</b>,<b>e</b>) cell smears and (<b>d</b>,<b>f</b>) the ex vivo cell preparations after (<b>c</b>,<b>d</b>) Romanovsky–Giemsa staining and (<b>e</b>,<b>f</b>) the immunofluorescence assay with specific antibodies to different NSCLC and fibroblast markers (green and red signals). Nuclei are stained by DAPI (blue signal). (<b>e</b>,<b>f</b>) Colocalization of the markers is (<b>e</b>) yellow and (<b>f</b>) magenta (in the nuclei) signals on confocal immunofluorescent images. The scale bars are (<b>c</b>,<b>d</b>) 10, (<b>e</b>) 20, and (<b>f</b>) 5 μm.</p> "> Figure 2
<p>Representative images after Romanovsky–Giemsa staining demonstrate that the differentiation and specific features of the patients’ adenocarcinoma cells and their clusters can be defined not only (<b>e</b>–<b>l</b>) on the histological sections, but also (<b>a</b>–<b>d</b>) on the ex vivo cell preparations obtained from the same tumor samples. (<b>e</b>–<b>h</b>) Close-ups of the parts of the images (<b>i</b>–<b>l</b>). The scale bars are (<b>i</b>) 5, (<b>a</b>–<b>d</b>,<b>j</b>–<b>l</b>) 10, and (<b>e</b>–<b>h</b>) 50 μm.</p> "> Figure 3
<p>Representative confocal merged immunofluorescent or immunochemical images demonstrate the expression of lung (<b>A</b>) adenocarcinoma- and (<b>B</b>) squamous-cell-carcinoma-specific markers and (<b>C</b>) proliferation marker Ki-67 in cancer cells both on the ex vivo cell preparations and, in parallel, on the histological sections obtained from the same tumor samples, while (<b>C</b>) PD-L1 expression is detected (<b>u</b>,<b>w</b>) in some lung squamous cell carcinoma cells only by ex vivo analysis. Cells and their nuclei are stained with appropriate specific antibodies (green and red signals or brown staining) and DAPI (blue signal), respectively. Localization of some markers in the nuclei is magenta signal. (<b>t</b>) Red arrow indicates the anaphase of mitosis. Green arrows indicate the PD-L1-positive cancer cells, as solitary and in clusters. The scale bars are (<b>a</b>,<b>b</b>,<b>h</b>,<b>j</b>,<b>q</b>,<b>t</b>) 5, (<b>d</b>,<b>f</b>,<b>g</b>,<b>k</b>–<b>p</b>,<b>r</b>,<b>w</b>) 10, and (<b>c</b>,<b>e</b>,<b>i</b>,<b>s</b>,<b>u</b>,<b>v</b>,<b>x</b>) 20 μm.</p> "> Figure 3 Cont.
<p>Representative confocal merged immunofluorescent or immunochemical images demonstrate the expression of lung (<b>A</b>) adenocarcinoma- and (<b>B</b>) squamous-cell-carcinoma-specific markers and (<b>C</b>) proliferation marker Ki-67 in cancer cells both on the ex vivo cell preparations and, in parallel, on the histological sections obtained from the same tumor samples, while (<b>C</b>) PD-L1 expression is detected (<b>u</b>,<b>w</b>) in some lung squamous cell carcinoma cells only by ex vivo analysis. Cells and their nuclei are stained with appropriate specific antibodies (green and red signals or brown staining) and DAPI (blue signal), respectively. Localization of some markers in the nuclei is magenta signal. (<b>t</b>) Red arrow indicates the anaphase of mitosis. Green arrows indicate the PD-L1-positive cancer cells, as solitary and in clusters. The scale bars are (<b>a</b>,<b>b</b>,<b>h</b>,<b>j</b>,<b>q</b>,<b>t</b>) 5, (<b>d</b>,<b>f</b>,<b>g</b>,<b>k</b>–<b>p</b>,<b>r</b>,<b>w</b>) 10, and (<b>c</b>,<b>e</b>,<b>i</b>,<b>s</b>,<b>u</b>,<b>v</b>,<b>x</b>) 20 μm.</p> "> Figure 4
<p>Representative images after Romanovsky–Giemsa staining demonstrate the different types of immune cells detected on the ex vivo cell preparations and, in parallel, on histological sections obtained from the same tumor samples (<b>a</b>–<b>l</b>) for tobacco smokers and (<b>m</b>–<b>x</b>) non-smoking patients. Red and green arrows indicate macrophages, as solitary and in clusters, with denser dark inclusions in the cytoplasm (smokers’ macrophages) and without them, respectively. Yellow and brown arrows indicate (<b>n</b>,<b>t</b>,<b>u</b>) neutrophils and (<b>b</b>,<b>e</b>) eosinophils, respectively, as solitary and in clusters. (<b>e</b>) The granules of eosinophils are visualized with DAB substrate. The scale bars are (<b>a</b>–<b>d</b>,<b>f</b>–<b>x</b>) 10 and (<b>e</b>) 50 μm.</p> "> Figure 5
<p>Differences in the number of immune cells in the TME between different NSCLC subtypes are found for the patients without tumor eosinophilia. The total number of immune cells (all types) is expressed as the percentage of the total number of the patients’ cells (cancer and immune) examined on the ex vivo cell preparations for adenocarcinoma ad1, ad3-ad8 (<span class="html-italic">n</span> = 7) and squamous cell carcinoma sq1, sq3, sq4 (<span class="html-italic">n</span> = 3) samples. Data are expressed as the means ± SEM. * <span class="html-italic">p</span> < 0.001.</p> "> Figure 6
<p>Most macrophages express all markers studied (<b>A</b>,<b>B</b>) in the tumor microenvironment of all patients’ samples and (<b>A</b>) in the lung tissue samples of the smoking NSCLC patients, whereas (<b>B</b>) alveolar macrophages only with CD14 expression are identified in the lung tissues for some non-smoking patients. The number of the marker-positive macrophages expressed as the percentage of the total number of the macrophages analyzed on the ex vivo cell preparations.</p> "> Figure 6 Cont.
<p>Most macrophages express all markers studied (<b>A</b>,<b>B</b>) in the tumor microenvironment of all patients’ samples and (<b>A</b>) in the lung tissue samples of the smoking NSCLC patients, whereas (<b>B</b>) alveolar macrophages only with CD14 expression are identified in the lung tissues for some non-smoking patients. The number of the marker-positive macrophages expressed as the percentage of the total number of the macrophages analyzed on the ex vivo cell preparations.</p> ">
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Patients, Their Tumor and Lung Tissue Samples
2.2. Ex Vivo Isolation of Cells and Production of Ex Vivo Cell Preparations
2.3. Cell Staining
2.4. Histology
2.5. Microscopy
2.6. Statistical Analysis
3. Results
3.1. Clinicopathological Characteristics of NSCLC Patients
3.2. Experimental Design and Cell Composition after Ex Vivo Isolation from Tumor Samples
3.3. Tumor-Related Markers Expressed by the Cancer Cells That Were Studied in Ex Vivo and Histological Analyses at Once
3.4. Immune Cell Landscape in the Tumor Microenvironment and Lung Tissue of NSCLC Patients
3.5. The Expression Pattern of Immune-Related Markers by Tumor-Associated Macrophages and Alveolar Macrophages
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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NSCLC Patients 1 | |||||||
---|---|---|---|---|---|---|---|
No. | Tumor Samples (g) 2 | TNM Classification | Sex 3 | Age (Years) | Smoking Status (Years) 4 | Attendant Pulmonology Diseases | Surgery 5 |
Adenocarcinoma | |||||||
ad1 | 1.06 | T1cN2A2M0(IIIA) | M | 72 | 45 | LLL | |
ad2 | 0.12 | T2aN0M0(IB) | F | 68 | 50 | COPD | RLL |
ad3 | 0.19 | T1cN0M0(IA3) | M | 62 | 50 | Chronic bronchitis | LUL |
ad4 | 0.20 | T1cN1M0(IIB) | M | 58 | 45 | RLL | |
ad5 | 0.17 | T2N0M0(IB) | M | 63 | 40 | LUL | |
ad6 | 0.04 | T2aN0M0(IB) | F | 55 | - | RUL | |
ad7 | 0.09 | T2bN0M0(IIA) | F | 64 | - | RLL | |
ad8 | 0.12 | T1bN0M0(IA2) | M | 63 | 54 | RLL | |
Squamous cell carcinoma | |||||||
sq1 | 0.07 | T1cN0M0(IA3) | M | 63 | 45 | Chronic bronchitis | RL |
sq2 | 0.13 | T1cN0M0(IA3) | M | 66 | 50 | LUL | |
sq3 | 0.12 | T3NxM0 | M | 67 | 40 (+), 10 (-) | RLL | |
sq4 | 0.12 | T1cN1M0(IIB) | M | 72 | 51 | COPD, pneumosclerosis | RUL |
NSCLC Markers 1 | NSCLC Patients | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Adenocarcinoma | Squamous Cell Carcinoma | |||||||||||
ad1 | ad2 | ad3 | ad4 | ad5 | ad6 | ad7 | ad8 | sq1 | sq2 | sq3 | sq4 | |
Lung adenocarcinoma-specific markers | ||||||||||||
CK7 | 5 | 1 | 1 | 1 | 10 | 5 | 2 | - | - | nd | nd | 0.5 |
TTF1 | - | 30 | 10 | - | - | - | - | - | - | nd | nd | - |
Lung squamous-cell-carcinoma-specific markers | ||||||||||||
PanCK | 5 | - | - | 5 | 10 | nd | nd | 0.5 | 0.5 | 1 | - | - |
p40 | nd | nd | nd | nd | nd | nd | nd | - | 30 | - | - | - |
Proliferation marker | ||||||||||||
Ki-67 | - | - | - | - | - | - | - | 10 | 50 | - | - | 20 |
Immunotherapy marker | ||||||||||||
PD-L1 | - | - | - | - | nd | - | - | - | 10 | - | 2 | - |
PD-L1 (histology) 2 | - | - | - | - | - | - | - | - | - | - | - | - |
Lung carcinogenesis markers | ||||||||||||
AhR | - | - | - | - | - | - | - | nd | - | - | - | nd |
AhRR | - | - | - | - | - | - | - | nd | nd | nd | nd | nd |
CYP1A1 | - | - | - | - | - | - | 10 | - | 10 | - | - | - |
Tobacco smoking 3 | + | + | + | + | + | - | - | + | + | + | - | + |
Immune cells 4 in the TME | ||||||||||||
Macrophages | 72.7 | 1.5 | 16.1 | 75 | 71.4 | 86.7 | 58.3 | 75 | 33.3 | 5.8 | 42.9 | 87.1 |
Neutrophils | 22.7 | 95.5 | 28.6 | - | 14.3 | 3.3 | 16.7 | - | 66.7 | 2.5 | 42.9 | - |
Lymphocytes | 4.6 | 3 | 1.2 | - | - | 10 | 25 | 25 | - | 0.8 | 14.3 | - |
Eosinophils | - | - | 54.2 | 25 | 28.6 | - | - | - | - | 90.9 | - | 12.9 |
Total number 5 | 7.4 | 41.2 | 18.4 | 11.8 | 13.8 | 14.2 | 12.4 | 3 | 0.9 | 28.6 | 0.3 | 2.4 |
Smokers’ macrophages | + | + | + | + | + | + | - | + | + | + | - | + |
Immune cells 4 in the lung tissue | ||||||||||||
Macrophages | 100 | 54 | 65.7 | 82.1 | 63.9 | 94 | 90.2 | 96.5 | 44.6 | 52.5 | 64.8 | 99 |
Neutrophils | - | 39.1 | 16.2 | 4.1 | 22.2 | 1.2 | 7 | 3.5 | 14.3 | 29.5 | 31.5 | - |
Lymphocytes | - | 6.9 | 18.2 | - | 12.5 | 4.8 | 2.8 | - | 41.1 | 11.5 | 3.7 | - |
Eosinophils | - | - | - | 13.8 | 1.4 | - | - | - | - | 6.6 | - | 1 |
Smokers’ macrophages | + | + | + | + | + | + | - | + | + | + | - | + |
Attendant immune diseases 3 | - | - | - | - | - | - | - | Asthma | - | - | - | - |
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Ufimtseva, E.G.; Gileva, M.S.; Kostenko, R.V.; Kozlov, V.V.; Gulyaeva, L.F. Development of Ex Vivo Analysis for Examining Cell Composition, Immunological Landscape, Tumor and Immune Related Markers in Non-Small-Cell Lung Cancer. Cancers 2024, 16, 2886. https://doi.org/10.3390/cancers16162886
Ufimtseva EG, Gileva MS, Kostenko RV, Kozlov VV, Gulyaeva LF. Development of Ex Vivo Analysis for Examining Cell Composition, Immunological Landscape, Tumor and Immune Related Markers in Non-Small-Cell Lung Cancer. Cancers. 2024; 16(16):2886. https://doi.org/10.3390/cancers16162886
Chicago/Turabian StyleUfimtseva, Elena G., Margarita S. Gileva, Ruslan V. Kostenko, Vadim V. Kozlov, and Lyudmila F. Gulyaeva. 2024. "Development of Ex Vivo Analysis for Examining Cell Composition, Immunological Landscape, Tumor and Immune Related Markers in Non-Small-Cell Lung Cancer" Cancers 16, no. 16: 2886. https://doi.org/10.3390/cancers16162886