Detailed Profiling of the Tumor Microenvironment in Ethnic Breast Cancer, Using Tissue Microarrays and Multiplex Immunofluorescence
Abstract
:1. Introduction
2. Results
2.1. Cohort Characteristics
2.2. Dominant Immune Cell Phenotypes in Different Regions of the Stained Cases
2.3. Distribution of Immune Cells in Relation to the Histopathologic Characteristics
2.3.1. Core Samples (Table S1 and Figure 2a)
2.3.2. TMA Samples (Table S2 and Figure 2b)
2.4. Statistical Correlation between TME Immune Cells, Histopathologucal Paramaters and Patient Outcome
2.4.1. Core Samples (Table S3 and Figure 3)
2.4.2. TMA Samples (Table S4 and Figure 4)
2.5. Summary of Findings (Figure S1)
2.6. Ethnic Groupings and Individual Ethnicities (Tables S1–S4 and Figure 2 and Figure S2)
3. Discussion
3.1. Th Cells
3.2. Treg Cells
3.3. Tc Cells
3.4. B Cells
3.5. TAMs
3.6. FOXP3-Positive Cells
3.7. Significant Findings
3.8. Strengths of the Study
3.9. Future Plans
4. Materials and Methods
4.1. Selection of Cases
4.2. Tissue Microarray (TMA) Construction
4.3. Multiplex Immunofluorescence (mIF)
4.4. Selecting Regions of Interest (ROIs) Using the Phenochart Software
4.5. Image Analysis Using InForm Software
4.6. Data Analysis Using Phenoptrreports Package in R Studio
4.7. Defining the Immune Cell Phenotypes
4.8. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Core Samples n = 87 | TMA Samples n = 143 | Patient Cohort n = 192 | |||||
---|---|---|---|---|---|---|---|
N | % | N | % | N | % | ||
Age at Diagnosis ‘Median (IQR)’ | 52 (44:59) | ||||||
Molecular Type | |||||||
Luminal | 27 | 31 | 116 | 81.1 | 124 | 64.6 | |
Her2-positive | 30 | 34.5 | 10 | 7 | 34 | 17.7 | |
TNBC | 30 | 34.5 | 17 | 11.9 | 34 | 17.7 | |
Tumor Grade | |||||||
Grade 1 | 0 | 0 | 20 | 14 | 20 | 10.4 | |
Grade 2 | 34 | 39 | 63 | 44 | 82 | 42.7 | |
Grade 3 | 53 | 61 | 60 | 42 | 90 | 46.9 | |
T. Size ‘Median (IQR)’ | 21 (15:28) | 19 (12:30) | 19 (14:28) | ||||
T-Stage | |||||||
T1 | 41 | 47.7 | 77 | 53.8 | 104 | 55.3 | |
T2 | 33 | 38.4 | 50 | 35 | 64 | 34 | |
T3 | 8 | 9.3 | 14 | 9.8 | 18 | 9.6 | |
T4 | 1 | 1.2 | 2 | 1.4 | 2 | 1.1 | |
N/A | 1 | 4 | |||||
Lymph nodes | |||||||
Nodal Status | |||||||
Positive | 30 | 34.5 | 52 | 36.4 | 71 | 37 | |
Negative | 57 | 65.5 | 91 | 63.6 | 121 | 63 | |
N-Stage | |||||||
N0 | 57 | 66.3 | 91 | 63.6 | 121 | 63.4 | |
N1 | 20 | 23.2 | 38 | 26.6 | 52 | 27.2 | |
N2 | 8 | 9.3 | 10 | 7 | 13 | 6.8 | |
N3 | 1 | 1.2 | 4 | 2.8 | 5 | 2.6 | |
N/A | 1 | 1 | |||||
Chemotherapy | |||||||
Received | 38 | 43.7 | 41 | 28.7 | 66 | 34.4 | |
Not Received | 49 | 56.3 | 102 | 71.3 | 126 | 56.6 | |
NPI * ‘Median (IQR)’ | 4.47 (3.52:5.16) | 4.3 (3.36:4.9) | 4.3 (3.37:4.92) | ||||
OS | ‘Median (IQR)’ | 105 (78:145) | 101 (84:117) | 103 (86.25:124:75) |
Ethnicity | Number | Ethnic Group | 5YS * | 10YS * |
---|---|---|---|---|
Pakistani | 66 (34.4%) | Asian n = 128 (66.6%) | 90.5% | 81% |
Indian | 42 (21.9%) | |||
Chinese | 6 (3.1%) | |||
Bangladeshi | 1 (0.5%) | |||
Asian (other) | 13 (6.8%) | |||
Caribbean | 24 (12.5%) | Black n = 46 (24%) | 85% | 71% |
African | 14 (7.3%) | |||
Black (other) | 8 (4.2%) | |||
Mixed | 8 (4.2%) | OTHER n = 18 (9.4%) | 83.5% | 73% |
Any other | 10 (5.2%) |
Immune Phenotype | Cores | TMA | |||
---|---|---|---|---|---|
N | % | N | % | ||
Th | Dominant | 62 | 71.3 | 99 | 69.2 |
2nd most predominant | 17 | 19.5 | 27 | 18.9 | |
Treg | Dominant | 0 | 0 | 0 | 0 |
2nd most predominant | 1 | 1.1 | 0 | 0 | |
Tc | Dominant | 13 | 14.9 | 9 | 6.3 |
2nd most predominant | 40 | 46 | 87 | 60.8 | |
B | Dominant | 5 | 5.7 | 27 | 18.9 |
2nd most predominant | 11 | 12.6 | 15 | 10.5 | |
TAM | Dominant | 6 | 6.9 | 8 | 5.6 |
2nd most predominant | 18 | 20.7 | 14 | 9.8 | |
FOXP3 | Dominant | 1 | 1.1 | 0 | 0 |
2nd most predominant | 0 | 0 | 0 | 0 |
Marker | Company | Clone | Host | Marker Dilution | Reporter | Reporter Dilution | Antigen Retrieval Method and Time (min) |
---|---|---|---|---|---|---|---|
CD20 | Leica | L26 | Mouse | 1:200 | Opal 480 | 1:150 | H2 (20) |
CD8 | Leica | 4B11 | Mouse | 1:400 | Opal 570 | 1:100 | H1 (20) |
CD4 | Abcam | EPR6855 | Rabbit | 1:200 | Opal 520 | 1:150 | H1 (20) |
FOXP3 | Abcam | 236A/E7 | Mouse | 1:400 | Opal 620 | 1:150 | H1 (20) |
CD68 | Leica | 514H12 | Mouse | 1:400 | Opal 690 | 1:150 | H1 (20) |
CK | Leica | AE1/AE3 | Mouse | 1:100 | Opal 780 | 1:10 | H1 (20) |
Filter | Duration (ms) |
---|---|
DAPI | 1.31 |
Cy5 | 8.26 |
FITC | 3.06 |
Cy3 | 16.95 |
Texas Red | 61.25 |
Opal 570 | 22.65 |
Opal 690 | 12.78 |
Cell Phenotype | Defined Staining Pattern |
---|---|
T-helper cells (Th) | CD4+/FOXP3- |
T-regulatory cells (Treg) | CD4+/FOXP3+ |
T-cytotoxic cells (Tc) | CD8+/FOXP3 |
B-cells | CD20+/FOXP3 |
Tumor-associated macrophages (TAMs) | CD68+/FOXP3 |
FOXP3-positive cells | FOXP3+/CD4-/CD8-/CD20-/CD68- |
IMM cells | Sum of Th, Treg, Tc, B, TAMs and FOXP3 |
ALL cells | Sum of IMM and DAPI+ cells (other TME cells) |
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Zaakouk, M.; Longworth, A.; Hunter, K.; Jiman, S.; Kearns, D.; El-Deftar, M.; Shaaban, A.M. Detailed Profiling of the Tumor Microenvironment in Ethnic Breast Cancer, Using Tissue Microarrays and Multiplex Immunofluorescence. Int. J. Mol. Sci. 2024, 25, 6501. https://doi.org/10.3390/ijms25126501
Zaakouk M, Longworth A, Hunter K, Jiman S, Kearns D, El-Deftar M, Shaaban AM. Detailed Profiling of the Tumor Microenvironment in Ethnic Breast Cancer, Using Tissue Microarrays and Multiplex Immunofluorescence. International Journal of Molecular Sciences. 2024; 25(12):6501. https://doi.org/10.3390/ijms25126501
Chicago/Turabian StyleZaakouk, Mohamed, Aisling Longworth, Kelly Hunter, Suhaib Jiman, Daniel Kearns, Mervat El-Deftar, and Abeer M Shaaban. 2024. "Detailed Profiling of the Tumor Microenvironment in Ethnic Breast Cancer, Using Tissue Microarrays and Multiplex Immunofluorescence" International Journal of Molecular Sciences 25, no. 12: 6501. https://doi.org/10.3390/ijms25126501