Ultrastructural Changes of the Peri-Tumoral Collagen Fibers and Fibrils Array in Different Stages of Mammary Cancer Progression

Abstract

Breast cancer invasion and subsequent metastasis to distant tissues occur when cancer cells lose cell-cell contact, develop a migrating phenotype, and invade the basement membrane (BM) and the extracellular matrix (ECM) to penetrate blood and lymphatic vessels. The identification of the mechanisms which induce the development from a ductal carcinoma in situ (DCIS) to a minimally invasive breast carcinoma (MIBC) is an emerging area of research in understanding tumor invasion and metastatic potential. To investigate the progression from DCIS to MIBC, we analyzed peritumoral collagen architecture using correlative scanning electron microscopy (SEM) on histological sections from human biopsies. In DCIS, the peritumoral collagen organizes into concentric lamellae ('circular fibers') parallel to the ducts. Within each lamella, type I collagen fibrils align in parallel, while neighboring lamellae show orthogonal fiber orientation. The concentric lamellar arrangement of collagen may physically constrain cancer cell migration, explaining the lack of visible tumor cell invasion into the peritumoral ECM in DCIS. A lamellar dissociation or the development of small inter fiber gaps allowed isolated breast cancer cell invasion and exosomes infiltration in the DCIS microenvironment. The radially arranged fibers observed in the peri-tumoral microenvironment of MIBC biopsies develop from a bending of the circular fibers of DCIS and drive a collective cancer cell invasion associated with an intense immune cell infiltrate. Type I collagen fibrils represent the peri-tumoral nano-environment which can play a mechanical role in regulating the development from DCIS to MIBC. Collectively, it is plausible to suggest that the ECM effectors implicated in breast cancer progression released by the interplay between cancer, stromal, and/or immune cells, and degrading inter fiber/fibril hydrophilic ECM components of the peritumoral ECM, may serve as key players in promoting the dissociation of the concentric collagen lamellae.

Keywords: breast cancer; collagen fibers; collagen fibrils; extracellular matrix.

Figure 1

Correlative SEM analysis of deparaffinized histological sections of DCIS and TACS-II stage human biopsies. (a) The hematoxylin-eosin histological section shows a transversally sectioned duct (D) including proliferating cancer cells which do not invade the ECM, as circular densely packed collagen fibers (C) running parallel to the BM which are able to oppose cancer cells migration. No large inter-fiber spaces are detectable very next to the duct and only a poor immune infiltrate is visible only far from the duct. Bar = 50 µm. (b) A particular region delimited by the rectangular box in the previous deparaffinized histological section analyzed by SEM shows proliferating polygonal breast cancer cells inside a duct (D), surrounded by circular collagen fibers (between the two arrows) including densely packed fibrils and running parallel to the duct surface. No gaps or nano spaces are detectable between the ductal cancer cells and collagen fibers. White bar = 10 µm. (c) Breast cancer human biopsy of DCIS corresponding to TACS-II. A small portion of a duct containing cancer cells (on the bottom right side of the picture) is surrounded by densely packed circular collagen fibers running parallel along the duct surface. Among the circular collagen fibers, very small fusiform gaps are visible. Bar = 50 µm. (d) A particular area delimited by the rectangular box in the previous deparaffinized histological section analized by SEM. Many breast cancer cells inside a duct (on the below right side) are confined by a circular collagen fiber composed of densely packed collagen fibrils (CFs) which strongly adhere to the cancer cell BM, but a small fusiform inter-fibrillar space 5X-12 µm wide (on the left) is detectable next to the duct. White bar = 10 µm.

Figure 2

SEM analysis of deparaffinized histological sections of DCIS and TACS II human biopsies. (a) The collagen lamellae adjacent to the duct surface (D) appear densely packed and contain bundled fibrils running in the same direction and parallel inside each lamella, but orthogonal vs. adjacent lamellae (arrows). White bar = 10 µm. (b) The circular collagen fibers (CFs) described in the literature as fibers surrounding and successfully confining breast cancer cells (CCs) correspond to circular concentric collagen lamellae containing fibrils showing an orthogonal array vs. adjacent lamellae. In this picture the single lamellae are separated by less than 10 µm-wide inter-fiber spaces. A small vessel containing three immune cells is visible on the left side of the picture. White bar = 10 µm. (c) At higher magnifications the orthogonal array of the fibrils in adjacent collagen lamellae (arrows) is evident. A duct is visible on the right side. White bar = 10 µm. (d) The circular or concentric lamellae surrounding a duct appear composed of collagen fibers separated by inter-lamellar spaces and include densely packed fibrils running longitudinally (L) or obliquely (O) vs. the duct axis. White bar = 1 µm.

Figure 3

Correlative microscopy by SEM analysis of deparaffinized histological sections of a DCIS and TACS-II stage human biopsy. Human biopsy corresponding to DCIS and TACS-II stage. (a) In the hematoxylin-eosin histological section, two adjacent ducts, completely filled with proliferating breast cancer cells, seem to fuse and appear surrounded by densely packed circular collagen lamellae (CL) and peri-tumoral desmoplastic reticular collagen meshwork (RM). No cancer cell invading the ECM is detectable. Bar = 50 µm. (b) A particular of the previous histological section, deparaffinized and analyzed by SEM, shows a portion of a duct containing a breast cancer cell (CC) shedding microvesicles which are confined by densely packed fibrils. Both the cancer cell and microvesicles are confined by densely packed circular fibrils which, adhering to the BM, seem to progressively substitute a thin (about 10 µm) layer of a fibril meshwork (between the two arrows) of large fibrils (100–120 nm) probably corresponding to initial TACS-I stage or desmoplasia. White bar = 10 µm. (c) The histological section shows a duct filled with densely packed proliferating cancer cells. The jamming ductal cancer cells protrude towards the ECM so that the epithelial basal line, surrounded by circular collagen lamellae, appears micro-mamillated. The black line delineates the ductal contour, following a wavy trajectory. Bar = 50 µm. (d) Small area of the (c) histological section, deparaffinized and analyzed by SEM. Ductal cancer cells show a rounded concave shape (large arrows) and the rounded empty spaces might correspond to detached or deeper cancer cells. Collagen fibers (small arrows) developing from the circular concentric CL penetrate the epithelial basal layer and surround the peripheral ductal cancer cells. Fusiform spaces are visible among the collagen fibrils. White bar = 10 µm.

Figure 4

Correlative microscopy by SEM analysis of deparaffinized histological sections of DCIS and TACS-II stage human biopsies. Human biopsy of DCIS and a TACS-II stage. (a) The histological section stained with Picro Sirius Red to evidence collagen observed under the polarized light microscope shows a portion of a duct (D) surrounded by circular but crimped densely buckled collagen lamellae which show inter-lamellar spaces/channels. Bar = 50 µm. (b) Ultrastructural observation of a deparaffinized histological section evidences the densely packed concentric collagen lamellae surrounding a duct filled with proliferating cancer cells and showing inter-lamellar spaces/channels due to a partial inter-fiber dissociation. The fibril orthogonal array in adjacent collagen lamellae is still observable (D). Bar = 10 µm. (c) A deparaffinized histological section analyzed by SEM shows that when the concentric collagen lamellae (on the right) in contact with cancer cells are not interrupted, the proliferating cancer cells (CCs), also displaying many microvesicles on their surface, are successfully confined. Bar = 10 µm. (d) Histological section stained with Picro Sirius Red observed under the polarized light microscope to evidence collagen fibers. The proliferating polygonal jamming cancer cells confined in a duct try to invade the concentric collagen lamellae which show an early partial disruption and dissociation (in the center of the picture). Bar = 50 µm. (e) By SEM analysis the CL next to the BM of the duct are partially interrupted and show a discontinuity which allowed exosomes (small arrows) and microvesicles (large arrows) to pass through and invade the ECM. A cancer cell covered and surrounded by many microvesicles (CC) is visible among the circular and concentric collagen lamellae. Bar = 10 µm. (f) In some areas of the same histological section observed with SEM, the CL seem to bend and dramatically change their direction assuming a radial array (very long arrows) vs. the duct surface (on the left) and corresponding to a TACS-III stage. Collagen fibers forming thin sheaths surround the cancer cells of the basal layer (*). Extravesicles are detectable inside an inter-fiber channel (arrow). Both longitudinally (top part of the picture) and transversally cut fibrils (bottom part of the picture) of the dissociated circular concentric collagen CL are visible in the ECM. Bar = 10 µm.

Figure 5

Correlative microscopy by SEM investigation of deparaffinized histological sections of a MIBC and TACS-II stage human biopsy. (a) In the hematoxylin–eosin histological section, the pressure of the jamming proliferating cancer cells completely filling the duct induces ECM invasion which favors an unjammimg state. A collective invasion of grouped cells is evident inside the area delimited by the blue line; the concentric peri-tumoral collagen lamellae show dissociation and inter-lamellar gaps in which both cancer cells and many immune cells are detectable. A conspicuous parvicellular infiltrate is present in inter-fiber spaces of the external collagen lamellae. Bar = 50 µm. (b) The previous histological section deparaffinized and evaluated by SEM shows that a collective ductal cancer cell invasion occurs where concentric collagen lamellae are separated by gaps filled with many rounded-shaped immune cells. Cytoplasmic processes similar to tunneling nanotubes (arrows) are evident among the adjacent rounded-shaped immune cells (presumably TAMs; upper arrow), as well as between immune and invading cancer cells (lower arrow). Bar = 10 µm. (c) Histological section stained with Picro Sirius Red to evidence collagen at the polarized light microscope. The jamming cancer cells inside the duct are invading the peri-tumoral ECM and show direct contact with the dissociated concentric collagen lamellae. Bar = 50 µm. (d) A particular area delimited by the rectangular box in the previous histological section deparaffinized and analyzed by SEM shows an invading ductal rounded cancer cell surrounded by and in tight contact with the dissociated collagen lamellae. The cancer cell develops short and ventral cytoplasmic processes similar to invadopodia (arrow). Bar = 10 µm.

Figure 6

Correlative microscopy by SEM analysis of deparaffinized histological sections of a MIBC and TACS-III stage human biopsy. (a) The histological section shows two mammary gland ducts (D) which are filled with proliferating cancer cells. Around the duct at the left side, densely packed and concentric collagen lamellae appear in continuity with straight and radially aligned collagen lamellae (very long arrows). Cancer cell invasion occurs in the gaps of dissociated concentric collagen lamellae, inside the blue line. An immune cell infiltrate is clearly observable around the invading cancer cells. Bar= 50 µm. (b) The same sample with Picro Sirius Red staining observed under the polarized light microscope shows grouped ductal cancer cells penetrating into the deep peri-tumoral ECM by crossing disrupted and randomly arranged collagen lamellae in red. Bar= 50 µm. (c) In a Picro Sirius Red stained section observed under the polarized light microscope, the concentric collagen lamellae surrounding small ducts are in continuity with straight radially aligned collagen fibers which form a wide conic track or channel for cancer cell invasion. Bar = 10 µm. (d) By SEM analysis the deparaffinized previous section demonstrates that the concentric collagen lamellae parallel to the surface of a duct (the right-hand side) change their direction, becoming radially aligned collagen lamellae (very long arrows). A cancer cell (small arrow) seems to penetrate along the inter-fiber channel. Bar = 10 µm. (e) The radially aligned collagen fibers (R) described in TACS-III stage could derive from the dissociation of the concentric collagen lamellae, which include circular (C), oblique (O), and longitudinal (L) fibers simply changing their direction. (f) A concave and rounded-shaped cancer cell develops invadopodia (arrow) and filopodia, which attach to the straight collagen fibrils of swollen fibers (on the left side) and thus begin to invade along narrow inter-fiber channels. Bar = 10 µm.

Figure 7

Correlative microscopy by SEM analysis of deparaffinized histological sections of a MIBC and TACS-III stage human biopsy. (a) The hematoxylin-eosin-stained histological section includes a portion of a duct (on the right) surrounded by CL which are not invaded by the cancer cells. However, in the outer layer of peri-tumoral ECM, dense bundles of collagen fibers following different directions are invaded by clusters of cancer cells (area inside the blue line), which seem to look for the straight outer radial fibers (on the left). Bar = 50 µm. (b) Histological section stained with Sirius Red and observed under the Polarized Light Microscope. Thin, straight, and aligned parallel collagen fibers which appear stained in red (arrows) delimit a cylindric channel for both cancer cell invasion (from the upside duct) and immune cell migration. Bar = 50 µm. (c) The deparaffinized section analyzed by SEM shows a massive infiltrate of immune cells floating among the straight and parallel collagen fibers. White bar = 10 µm. (d) In the same deparaffinized section observed with SEM, a polygonal invading cancer cell (CC) is penetrating between parallel, thin, and straight radially arranged fibers (arrows). The cancer cell strongly adheres to collagen fibers but also looks for adhesion to collagen by developing cytoplasmic protrusions (on its left side). Bar = 10 µm. (e) Two polygonal single cancer cells invading the peri-tumoral ECM intimately adhere to the collagen lamellae composed of collagen fibers, show extravesicles on their cytoplasmic surface, and develop invadopodia (arrow). White bar = 10 µm. (f) By SEM analysis two single and thin collagen fibers (CFs) including very densely packed collagen fibrils delimit a long inter-fiber channel (about 10 µm wide) containing polygonal-shaped cancer cells which produce extravesicles, display cell–cell contact and stick to the collagen wall of the channel. White bar = 10 µm. (g) By SEM analysis an inter-fiber channel transversally cut contains two cancer cells (CCs) producing and releasing extravesicles. Note a single microvesicle in a relative space inside the collagen fibers (arrow). White bar = 10 µm. (h) A deparaffinized section observed by SEM shows that very dense collagen fibers of ECM, far from the ducts, do not show the single fibrils they are composed of, and confine inter-fiber channels which contain cancer cells (downside) and three immune cells (upside). White bar = 10 µm. (i) By SEM analysis a single rounded-shaped immune cell almost completely encapsulated and covered by dense collagen fibers (in the red circle) is degrading and invading the peri-tumoral collagen ECM, following the arrow direction. An empty hole or inter-fiber channel in ECM is visible below. Black Bar = 10 µm. (j) In a deparaffinized section analyzed by SEM, a single immune cell strongly adhering to the wall of an inter-fiber channel develops cytoplasmic protrusions to migrate into a preformed ECM channel. Far from the cell, a few isolated microvesicles are also visible. White bar = 10 µm.

Figure 8

Dynamic changes in collagen fibril organization during breast cancer progression. The transition from ductal carcinoma in situ (DCIS) to microinvasive breast carcinoma (MIBC) involves the profound remodeling of the tumor microenvironment (TME), particularly the peri-tumoral collagen architecture. In the early stages, epithelial cells of the primary tumor are surrounded by concentric collagen lamellae composed of densely packed fibrils arranged orthogonally across adjacent layers. This organization generates a biophysical barrier that resists tumor expansion via tension forces and structural compaction. Tumor-derived paracrine signals trigger a desmoplastic reaction, leading to the activation of resident fibroblasts into cancer-associated fibroblasts (CAFs), which promote collagen cross-linking mainly through the lysyl oxidase (LOX) function, increase matrix stiffness, and alter local charge distributions through enzymatic remodeling by matrix proteolytic enzymes, such as metalloproteinases (MMPs). Concurrently, tumor-associated macrophages (TAMs) infiltrate the stroma and contribute to collagen degradation and reorganization. These immune and stromal interactions reshape the hydration state of the extracellular matrix (ECM), as proteoglycans (PGs) and glycosaminoglycans (GAGs), mainly hyaluronan (HA), modulate water content, viscoelasticity, and osmotic pressure that influence both cell motility and matrix mechanosensing. As invasive cancer cells invade the basement membrane (BM), they encounter a mechanically and chemically altered stroma facilitating the alteration in the provisional matrix and the formation of a pre-metastatic niche. Collagen fibers adjacent to these cells display tumor-associated collagen signatures (TACS). In TACS-II, collagen fibers align parallel to the tumor border, creating physical barriers; in TACS-III, they become linearized and oriented perpendicularly, forming channels that guide cancer cell migration/invasion and the initiation of metastasis. These structural changes are influenced by local tension forces, changes in charge density of matrix macromolecules, and hydration-dependent phenomena of interfibrillar spaces. These microenvironmental alterations not only facilitate cancer cell invasion but also shape immune cell infiltration and extracellular vesicle (EV) secretion, thereby reinforcing breast cancer progression.