Fig 1: Histotopographic features of the distribution of CPA3 + mast cells in organs. Primary antibodies used: Rabbit polyclonal to CPA3 antibody (ab251696) (AbCam). Secondary antibodies used: AmpliStain anti-Rabbit 1-Step HRP (#AS-R1-HRP), SDT GmbH, label-HRP. (a) Tonsil. Localization of CPA3+ MC in the lymphoid tissue (indicated by the arrow) and stroma (double arrow) of the organ. (b,c) The jejunum. The location of CPA3+ MC in the mucosa (indicated by the arrow) and submucosa (indicated by the double arrow). (d) Tonsilla. CPA3+ mast cell in interfollicular lymphoid tissue. (e–g) Jejunum. CPA3+ MC contacting in the lamina propria of the mucous (e), interaction with stromal cells (indicated by an arrow) and epithelium (indicated by a double arrow) of the mucous (f,g). (h–i) Stomach. CPA3+ MC in the stroma of the lamina propria of mucosa, colocalization with the parietal cell of the fundic gland (arrow) and stromal cells (h). CPA3+ MC degranulates in the direction of the perineurium (indicated by the arrow) and the lymphocyte (double arrow) of the submucosa (i). (j,k) Skin. Histotopographically, CPA3+ MC is colocalized with two capillaries (indicated by an arrow); however, CPA3 + secretory material selectively surrounds only one of them over a large area of the basement membrane of the endothelium (indicated by a double arrow) (j). MC is colocalized with stromal cells; it is obvious that CPA3 can affect several cells simultaneously, including fibroblast (indicated by an arrow) and an immunocompetent cell (k). (l) Small intestine. The location of CPA3+ MC near the macrophage (indicated by the arrow). (m) Small intestine. Interaction of CPA3+ MC and an activated lymphocyte (indicated by an arrow). (n) Skin. CPA3 + MC in the tumor microenvironment of melanoma contacts with immunocompetent cells and fibroblast (indicated by an arrow). (o–q) Melanoma of the skin. Various variants of colocalization of CPA3+ MC with fibroblasts of the stromal component (indicated by an arrow). Directed exocytosis of CPA3+ secretory granules (o), as well as morphological evidence of transgranulation, «kiss-and-run» degranulation and formation of exosomes (possibly) for the secretion of CPA3 to biological targets (p–q) are determined. (r–s) Skin melanoma. Active participation of CPA3 + MC in remodeling of the fibrous (r) and amorphous (s) components of the extracellular matrix of the tumor microenvironment (indicated by the arrow). Scale bar: 100 μm (a–c), 5 μm (the rest).
Fig 2: Morphological equivalents of mast cell CPA3 secretory pathways. Primary antibodies used: Rabbit polyclonal to CPA3 antibody (ab251696) (a–u) (AbCam), and Mouse monoclonal [NK1/C3] to CD63 antibody (ab1318) (f) (AbCam). Secondary antibodies used: (a–c,g–l,n–q,s,t) AmpliStain anti-Rabbit 1-Step HRP (#AS-R1-HRP), SDT GmbH, Baesweiler, Germany, label-HRP. (d–f,m,r,u) Goat anti-rabbit IgG Ab (#A-11034) Invitrogen, label-Cy3. (f) Goat anti-mouse IgG Ab (#A-11029), Invitrogen, label Alexa Fluor 488. (a,b) Tonsil. Compound CPA3 exocytosis with probable involvement of piecemeal degranulation. Selective accumulation of CPA3 in targets of a specific tissue microenvironment (indicated by an arrow) (a), accumulation of protease in the pericellular space of the extracellular matrix (indicated by an arrow) (b). (c) Tonsil. Formation of MC loci with the most intense CPA3 secretion (indicated by an arrow). (d,e) Skin. Secretion of CPA3 into the extracellular matrix by the mechanism of compound exocytosis and piecemeal degranulation, with the accumulation of protease in the pericellular space of neighboring cells (indicated by an arrow) (d). Formation of a separate MC cytoplasmic locus for CPA3 secretion (indicated by an arrow) (e). (f) Melanoma of the skin. High content of exosomes in CPA3+ mast cell. (g,r) Morphological variants of targeted degranulation of CPA3 by the mechanism of exocytosis to cellular targets in the specific tissue microenvironment of the tonsilla (g–j) and skin (k–m) (indicated by an arrow). (n–p) Tonsil. The initial stages of mast cell degranulation by the mechanism of exocytosis (n,o) (indicated by an arrow), leading to the distribution of CPA3 + secretory granules in the extracellular matrix (p) (indicated by an arrow). (q–r) Skin. Active exocytosis of mature secretory MC granules into the extracellular matrix with the formation of loci of the tissue microenvironment with a high content of CPA3+ granules (indicated by an arrow). (s–u) skin process of formation of CPA3+ macrovesicles of small (s) and large sizes (t,u) (indicated by an arrow). Scale bar: 5 μm.
Fig 3: Targeted effect of specific proteases on cells of the tumor microenvironment in melanoma. (a) Combined intense targeted secretion of tryptase and CPA3 to an adjacent cell over a large area of influence (indicated by an arrow). (b) Release of secretory granules with the tryptase+CPA3+ phenotype; some of them reach the nucleus of the adjacent cell (indicated by an arrow). (c) Formation of a locus of tryptase and CPA3 directed secretion to a cell of the tumor microenvironment (indicated by an arrow). (d) Adjacency of two MCs to the target cell with selective exocytosis of tryptase into the nuclear region (indicated by an arrow). (e,f) Variants of the formation of the MC cytoplasm elongated section towards the target cells (indicated by an arrow). Scale bar: 5 µm.
Fig 4: Protease phenotype and sectoral activity of MCs during the formation of macrovesicles and cytoplasts in the tumor microenvironment. (a) Eccentric position of the nucleus in MCs with the expression of a triad of specific proteases, with signs of detachment of a large cytoplasm fragment from one of them (indicated by an arrow). (b) Signs of the start of the nucleus release and MC enucleation (indicated by an arrow). (c) Colocalization of protease-positive MCs with a nuclear-free backbone of the cytoplasm with signs of secretory activity (indicated by an arrow). (d) Enucleation of the nucleus is accompanied by selective secretion as part of CPA3 granules (indicated by an arrow), or together with tryptase (indicated by a double arrow). (e) Completion of the nucleus exit from the MC with the formation of a cavity in the cytoplasm (indicated by an arrow). (f–h) The terminal stage of MC enucleation with the formation of a cytoplasmic backbone and preservation of the stock of specific proteases in the composition of the granules and secretory activity (f), indicated by an arrow). (g,h) Splitting the mother cytoplasm into two smaller fragments, the granules have a variability in the content of specific proteases, including granules with predominant accumulation of CPA3 (indicated by an asterisk) and tryptase (indicated by a double arrow). Scale bar: 5 µm.
Fig 5: Morphological equivalents of secretory pathways of specific MC proteases in the tumor microenvironment. (a) Different phenotype of specific proteases of secretory granules in the intercellular substance after secretion: tryptase+ (red arrow), chymase+ (green arrow), tipase+chymase+ (white arrow), chymase+tryptase+CPA3+ (double arrow). (a’) The same as (a) at higher magnification. (b) Simultaneous secretion of tryptase combined with CPA3 (indicated by an arrow), and chymase (indicated by a double arrow). (c,d) High degree of immunopositivity to CD63 of chymase-containing and tryptase-containing MCs. Secretory granules differ in the degree of immunopositivity for CD63 in the extracellular matrix (c’) and MC cytoplasm (d’), including the absence (indicated by the arrow). (e,f) LAMP1 expression in CPA3+ mast cell (e) and chymase+ MCs (f).
Supplier Page from Abcam for Anti-CPA3 antibody