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. 2023 Dec 21:13:1281650.
doi: 10.3389/fonc.2023.1281650. eCollection 2023.

Exploration of cancer associated fibroblasts phenotypes in the tumor microenvironment of classical and pleomorphic Invasive Lobular Carcinoma

Harsh Batra  1 , Qingqing Ding  2 , Renganayaki Pandurengan  1 , Heladio Ibarguen  1 , Neus Bota Rabassedas  1 , Aysegul Sahin  2 , Ignacio Wistuba  1 , Edwin Roger Parra  1 , Maria Gabriela Raso  1
Affiliations

Exploration of cancer associated fibroblasts phenotypes in the tumor microenvironment of classical and pleomorphic Invasive Lobular Carcinoma

Harsh Batra et al. Front Oncol. .

Abstract

As the second most common subtype of breast carcinoma, Invasive Lobular Carcinoma (ILC) microenvironment features have not been thoroughly explored. ILC has different histological subtypes and elucidating differences in their microenvironments could lead to a comprehensive development of cancer therapies. We designed a custom-made cancer associated fibroblast (CAFs) panel and used multiplex immunofluorescence to identify the differences in tumor microenvironment between Classic ILC and Pleomorphic ILC.

Materials and methods: Multiplex immunofluorescence were performed on formalin fixed paraffin embedded tissues using Opal-7 color kit. The antibodies used for phenotyping CAFs were Pan CK (AE1/AE3), CD45, A-SMA, FAP, S100, Thy-1 with optimized dilutions. The images were acquired and analyzed using Vectra 3.0 imaging system and InForm software respectively.

Results: We studied 19 different CAFs colocalized phenotypes in the tumor, stroma and overall tissue compartments between classic and pleomorphic ILC. Total A-SMA+, A-SMA+FAP+S100+ and A-SMA+S100+ CAFs demonstrated higher densities in classic ILC cases while FAP+S100+ and S-100+ CAFs were increased in the pleomorphic subtype samples.

Conclusion: Our study explores multiple CAFs phenotypes between classical and pleomorphic ILC. We showed that CAFs subset differ between Classic ILC and Pleomorphic ILC. A-SMA CAFs are more prevalent in the TME of classic ILCs whereas Pleomorphic ILCs are dominated by CAFs without A-SMA expression. This also iterates the importance of exploring this particular type of breast carcinoma in more detail, paving the way for meaningful translational research.

Keywords: Computational Pathology; ILC; Invasive Lobular Carcinoma; Multiplex immunofluorescence; cancer associate fibroblasts; tumor micro environment (TME).

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
(A) [(i) & (ii)] shows two cases of classic ILC(H&E). (B) [(iii) & (iv)] shows high magnification images of two cases of pleomorphic ILC (H&E).
Figure 2
Figure 2
Staining Pattern of different markers in the panel: Thy-1 (orange), FAP+ CAFs (Yellow), A-SMA CAFs (Pink) and S-100 CAFs (Green). The Tumor cells which are CK+ are colored cyan (Centre).
Figure 3
Figure 3
(A) CAF Phenotypes which were higher in density (statistically significant) in the TME of pleomorphic ILC as compared to classic ILC: (i) Distribution of FAP+S-100 (n/mm^2) by tumor types in stroma compartment; ii) Distribution of Total FAP (n/mm^2) by tumor types in tumor compartment ; iii) Distribution of S-100 only (n/mm^2) by tumor types in total compartment. (B) CAF Phenotypes which were higher in density the TME of classic ILC as compared to pleomorphic ILC: (i) Distribution of Total A-SMA (n/mm^2) by tumor types in total Compartment ; (ii) Distribution of A-SMA+S-100+ (n/mm^2 by tumor types in total Compartment; (iii) Distribution of A-SMA+FAP+S-100 (n/mm^2) by tumor types in total Compartment.
Figure 4
Figure 4
(A) Cases showing comparison of A-SMA densities (pink) between pleomorphic ILC (left) and classic ILC (right). The CK+ cells are tumor cells in cyan color. The classic ILC shows a higher density of A-SMA CAFs as compared to pleomorphic ILC. (B) Cases showing comparison of S-100 densities (green) between pleomorphic ILC (left) and classic ILC (right). (Tumor cells in cyan). The pleomorphic ILC shows a higher density of S-100 CAFs as compared to classic ILC. (C) Cases showing comparison of A-SMA(pink)+S-100(green) co-positive CAF densities between pleomorphic ILC (top-left) and classic ILC (top-right). The CAFs in the classic ILC (box in upper right image) are magnified in the bottom row and shows individual positivity for Alpha SMA (Lower left image) and S100 (lower right image). Note: The A-SMA positive (pink) structures in the pleomorphic ILC are blood vessels and were excluded for data interpretation. (D) Cases showing comparison of A-SMA(pink)+FAP(yellow)+S-100(green) copositive CAF densities between pleomorphic ILC and classic ILC (Center panel-Top row). The center panel-bottom row shows absence of Alpha-SMA positivity in CAFs in the pleomorphic ILC case {(the positivity of A-SMA is in the blood vessels (three of them are highlighted in boxes)as examples, not CAFs. The CAFs are either positive for FAP(yellow) or S100 (green) singly or in copositivity}. The side images show high power view of the same for pleomorphic ILC (Left side panel) and classic ILC ( Right side panel).
Figure 5
Figure 5
(A) The figure shows statistically significant differences in the proximity of CAFs from tumor cells (A-SMA+ and A-SMA+S100+) between Classic ILC and Pleomorphic ILC. (X-axis: tumor type; Y-axis: distance of CAFs from Tumor cells). The A-SMA+ and A-SMA+S100+ CAFs are nearer to tumor cells in Classic ILC as compared to Pleomorphic ILC. (B) The figure shows statistically significant differences in the proximity of CAFs from tumor cells (A-SMA-/CK-/FAP-/CD45-/S100+/Thy1-) between Classic ILC and Pleomorphic ILC. (X-axis: tumor type; Y-axis: distance of CAFs from Tumor cells). The A-SMA-/CK-/FAP-/CD45-/S100+/Thy1- CAFs are nearer to tumor cells in Pleomorphic ILC as compared to Classic ILC.
Figure 6
Figure 6
(A) Proximity of A-SMA positive CAFs (purple) to tumor cells (cyan) in classic ILC vs pleomorphic ILC. A-SMA+ CAFs are neare to the tumor cells in Classic ILC as compared to Pleomorphic ILC. (B) Proximity of S-100 positive CAFs (green) to tumor cells (cyan) in classic ILC (left) vs pleomorphic ILC (right). The S-100+ CAFs are closer to the tumor cells in Pleomorphic ILC as compared to the Classic ILC.
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