CD34+ Stromal Cells/Telocytes as a Source of Cancer-Associated Fibroblasts (CAFs) in Invasive Lobular Carcinoma of the Breast

Abstract

Several origins have been proposed for cancer-associated fibroblasts (CAFs), including resident CD34+ stromal cells/telocytes (CD34+SCs/TCs). The characteristics and arrangement of mammary CD34+SCs/TCs are well known and invasive lobular carcinoma of the breast (ILC) is one of the few malignant epithelial tumours with stromal cells that can express CD34 or αSMA, which could facilitate tracking these cells. Our objective is to assess whether tissue-resident CD34+SCs/TCs participate in the origin of CAFs in ILCs. For this purpose, using conventional and immunohistochemical procedures, we studied stromal cells in ILCs (n:42) and in normal breasts (n:6, also using electron microscopy). The results showed (a) the presence of anti-CD34+ or anti-αSMA+ stromal cells in varying proportion (from very rare in one of the markers to balanced) around nests/strands of neoplastic cells, (b) a similar arrangement and location of stromal cells in ILC to CD34+SCs/TCs in the normal breast, (c) both types of stromal cells coinciding around the same nest of neoplastic cells and (d) the coexpression of CD34 and αSMA in stromal cells in ILC. In conclusion, our findings support the hypothesis that resident CD34+SCs/TCs participate as an important source of CAFs in ILC. Further studies are required in this regard in other tumours.

Keywords: CAFs; CD34+ stromal cells; breast; cancer-associated fibroblasts; invasive lobular carcinoma; telocytes.

Figure 1

Stromal cells around nests of neoplastic cells in invasive lobular carcinoma of the breast. Double immunochemistry for CD34 (brown) and αSMA (red). Haematoxylin counterstain. Inserts: Double immunofluorescence labelling for CD34 (green) and αSMA (red) with DAPI (blue) counterstain. The stromal cells surrounding the neoplastic nests express CD34 (brown) in (A) and αSMA (red) in (B). In (C), the stromal cells express CD34 or αSMA, depending on the nest of neoplastic cells around which they are located. Note part of a duct in A, in which myoepithelial cells express αSMA (arrow). In B, a small vessel in which ECs express CD34 (arrow). The inserts show similar results by double immunofluorescence. Bar: (A): 15 µm; (B): 10 µm; (C): 8 µm.

Figure 2

(A) and inserts: Double immunochemistry staining for CD34 (brown) and αSMA (red). Haematoxylin counterstain. Vessels (V) and stromal cells (arrows) are observed around neoplastic cells (Haematoxylin-stained nuclei). The expression of CD34 is more intense in ECs than in stromal cells. Pericytes (arrowhead) are observed around ECs (insert 1 and 2). (B,C): Double immunofluorescence labelling for CD34 (green) and CD31 (red) with DAPI counterstain. Vessels (arrows), stromal cells (green) and nuclei of neoplastic cells are observed. Note CD34/CD31 coexpression in vessel ECs, while the stromal cells only express CD34. (D,E): Double immunochemistry staining for CD34 (brown) and Cytokeratin AE1/AE3 (red). Haematoxylin counterstain. Presence of neoplastic cells immunostained with anti-cytokeratin AE1/AE3 (red), surrounded by CD34+ stromal cells (brown). Bar: (A,D,E): 10 µm; (B,C): 30 µm; Insert 1 and 2 of (A): 20 µm.

Figure 3

(A,B,E,F): Double immunochemistry for CD34 (brown) and αSMA (red). Haematoxylin counterstain. (C,D): Double immunofluorescence labelling for CD34 (green) and αSMA (red). DAPI counterstain. (G,H): Ultrathin sections. Uranyl acetate and Lead citrate. (A–D): Processes of αSMA+ stromal cells (red) forming veils (arrows) that cover neoplastic cells (blue stained nuclei) (in (A,B), sections parallel to the surface of the processes; in (C,D), individual visions at different heights in confocal microscopy, using tissue sections of 10 µm). (E): Several layers of CD34+SCs/TCs (brown) around a normal breast duct in which the external layer of myoepithelial cells expresses αSMA (red). (F): One CD34+SC (brown, arrow) is observed around myoepithelial cells (red) of a component of the terminal ductal-lobular unit. (G,H): Ultrastructural images of long, thin telocyte extensions (telopodes) with podomeres (H arrows) and podoms (arrowhead) around ductal myoepithelial cells. Insert of (G) shows a contact between telopodes (arrowhead). Bar: (A–D): 8 µm; (E): 30 µm; (F): 20 µm; (G,H): 4 µm.

Figure 4

(A): Immunochemistry staining for CD34. Haematoxylin counterstain. Rings of CD34+SCs (brown) surrounding neoplastic cells around a glandular duct. Insert: a duct with the peripheral rings of neoplastic cells, which is virtually diagnostic of invasive lobular carcinoma in Haematoxylin-eosin staining. (B): Double immunochemistry staining for CD34 (brown) and αSMA (red). Haematoxylin counterstain. Abundant CD34+ (brown) and scarce SMA+ (red, arrows) stromal cells around a duct with αSMA+ myoepithelial cells. Insert: Double immunochemistry staining for CD34 (brown) and cytokeratin AE1/AE3 (red). Epithelial cells of the duct and neoplastic cells express cytokeratin AE1/AE3 (red) and stromal cells express CD34 (brown). Bar: (A,B): 30 µm; Insert of (A): 120 µm and Insert of (B): 80 µm.

Figure 5

Double immunofluorescence labelling for CD34 (green) and αSMA (red) with DAPI (blue) counterstain. (A): CD34+SCs/TCs (green) around a duct. (B,C): Rings of CD34+ (green) and αSMA+ (red) stromal cells surrounding neoplastic cells (blue stained nuclei) around ducts. Note the predominance of CD34+ stromal cells in (B) and of αSMA+ stromal cells in (C). Some stromal cells coexpress both markers. Bar: (A): 40 µm; (B,C): 30 µm.

Figure 6

Double immunochemistry for CD34 (brown) and αSMA (red). Haematoxylin counterstain. (A): Part of a lobule in which CD34+SCs/TCs are observed in the interstitium. (B): CD34+ stromal cells surrounding neoplastic cells in the interstitium of a lobule. (C): Several layers of CD34+SCs/TCs are seen in the adventitia of an artery. The insert shows a similar image in double immunofluorescence labelling. (D): CD34+ stromal cells (arrows) and occasional αSMA+ cells (arrowhead) around neoplastic cells in the adventitia of an artery. The insert shows positivity for CD34 (arrow) and αSMA (arrowheads) in stromal cells. Bar: (A,B,D): 30 µm; C: 15 µm; Insert of (C,D): 60 µm.

Figure 7

(A,B,D–F): Double immunochemistry staining for CD34 (brown) and αSMA (red). Haematoxylin counterstain. (C): Ultrathin section. Uranyl acetate and Lead citrate. (A): One CD34+SC/TC (arrow) around a small vessel. (B): Stromal cell processes (arrows) are observed surrounding neoplastic cells around a small vessel. (C): Ultrastructural image of telocyte telopodes (arrows) between collagen (CO) components in the interlobular connective tissue. (D): One CD34+ stromal cell with long processes, one of which surrounds a neoplastic cell. (E): CD34+SCs/TCs (arrows) between adipocytes (ad). (F): CD34+ (brown) and αSMA+ (red) stromal cells (arrows) are seen around a nest of neoplastic cells between adipocytes (ad). Bar: (A): 30 µm; (B,F): 20 µm; (C): 4 µm; (D,E): 15 µm.

Figure 8

Double immunochemistry labelling for CD34 (brown) and αSMA (red). Haematoxylin counterstain. (A,B): CD34+SCs (brown) around neoplastic cells in the smooth muscle fascicles (red) of the breast nipple. (C): αSMA+ stromal cells (red) in the epi-perineurium of a small nerve surrounding neoplastic cells (arrow). Note CD34+SCs/TCs (brown, arrowheads) in the endoneurium. (D,E): CD34+ (brown) and αSMA+ (red) stromal cells around the same nest (D) and strand (E) of neoplastic cells. Bar: (A): 30 µm; (B,D): 10 µm; (C) 25 µm; (E): 10 µm.

Figure 9

Double immunofluorescence labelling for CD34 (green) and αSMA (red) with DAPI (blue) counterstain. Coexpression of CD34 and αSMA is observed in stromal cells around neoplastic cells. Note cells expressing CD34 (green, (A)1, (B)1, (C)1 and (D)1), αSMA (red, (A)2, (B)2, (C)2 and (D)2) and both markers merged (yellow, (A)3, (B)3, (C)3 and (D)3). Bar: (A,B,D): 10 µm; (C): 20 µm.

Figure 10

Double immunofluorescence labelling for CD34 (green) and αSMA (red) with DAPI (blue) counterstain. Numerous cells expressing both markers are observed where CD34+SCs predominate (A) or where there is a balanced proportion of CD34+ and αSMA+ stromal cells (B) rather than where αSMA+ stromal cells predominate (C). Numbers indicate the expression of CD34 (1), αSMA (2) and merged expression of CD34 and αSMA (3). Bar: (A–C): 20 µm.