Analysis of tumor stem cell recruitment and integration in microvascular architectural heterogeneity during oral mucosal carcinogenesis: an in vivo experimental study

Figures

• 

  Fig. 1. Construction of an animal disease model for the oral mucosal carcinogenesis. (A) Gross view of the animal disease model for oral mucosal carcinogenesis and pathological characterization observed by HE staining (n = 10). (B) From left to right, images show immunofluorescence staining of CD44 and CD90. CD44 and CD90 were selectively and highly expressed in some stem cell subsets of OSCC tumor cells, showing red fluorescence. The MMI CellCut system was used to identify HSC4 clusters with positive CD44 and CD90 expression. The laser precisely cut the target cells along the marked line, separating the MMI membrane ring from the MMI isolation dish. The CD44- and CD90-positive cell clusters captured by laser were collected in the MMI isolation dish to obtain TSCs (TRITC, ×100, n = 6). (C) Single cells were screened using the limiting dilution method. After continuous culture for 18 days under stem cell culture conditions, TSCs clusters with a defined density were obtained (inverted microscope, ×100, n = 6). (D) Gross view of the animal disease model for oral mucosal carcinogenesis with TSCs interference and pathological characterization observed by HE staining (n = 10). (E) HE staining…

• 

  Fig. 2. EdU in vivo tracing of TSCs in organ microvessels. (A) After TSCs labeled with EdU Apollo 567 were injected into nude mice via the tail vein, they were distributed throughout multiple organs. During cancer initiation and progression, TSCs tended to migrate to tumor tissue, where they grew and proliferated, showing red fluorescence, whereas normal and non-TSC regions showed blue fluorescence (EdU, ×400, n = 6). (B) EdU in vivo tracing of TSCs in the egn, egpl, eghdscc, egmdscc, and egpdscc groups. After TSCs labeled with EdU Apollo 567 were injected into nude mice via the tail vein, they were distributed across multiple organs. As cancer progressed, TSCs tended to localize around microvessels in the heart, liver, spleen, lungs, and kidneys, showing red fluorescence, whereas normal and non-TSC regions showed blue fluorescence (EdU, ×400, n = 6). TSC, tumor stem cell

• 

  Fig. 3. Classical representations of microvascular morphological architecture heterogeneity in the oral mucosal carcinogenesis. (A) Classical representations of microvascular morphological architecture heterogeneity in the animal disease model of oral mucosal carcinogenesis were classified into terminal expansion, crisscross, finger-shaped propulsion, budding, and fusion types (HE, ×200, n = 6). (B) LCM was used to capture the classical representations of microvascular morphological architecture heterogeneity in the control groups cgn, cgpl, cghdscc, cgmdscc, and cgpdscc (HE, ×200, n = 6). (C) LCM was used to capture the classical representations of microvascular morphological architecture heterogeneity in the experimental groups egn, egpl, eghdscc, egmdscc, and egpdscc (HE, ×200, n = 6). HE, hematoxylin and eosin; LCM, laser capture microdissection

• 

  Fig. 4. LCM-targeted capture of representative microvascular morphological heterogeneity across oral mucosal carcinogenesis stages. (A) LCM-targeted capture of representative vascular morphologies in cgn, cgpl, cghdscc, cgmdscc, and cgpdscc (HE, ×200, n = 6). (B) LCM-targeted capture of representative vascular morphologies in egn, egpl, eghdscc, egmdscc, and egpdscc (HE, ×200, n = 6). HE, hematoxylin and eosin; LCM, laser capture microdissection

• 

  Fig. 5. TMT analysis of microvascular tissues with the dominant morphology of microvascular morphological architecture heterogeneity. (A) PCA plot comparing experimental and control samples during oral mucosal carcinogenesis. (B) Clustering plot (Protein C) illustrating sample grouping during oral mucosal carcinogenesis. (C) Venn diagram of upregulated differentially expressed proteins between the experimental and control groups. (D) Venn diagram of downregulated differentially expressed proteins between the experimental and control groups. (E) UpSet plot showing differentially expressed proteins between the control group and the upregulated and downregulated proteins. (F) UpSet plot showing differentially expressed proteins between the experimental group and the upregulated and downregulated proteins. TMT, tandem mass tag; PCA, principal component analysis

• 

  Fig. 6. Differential expression analysis of ANGPTL2, CK15, and EEF1E1 in the oral mucosal carcinogenesis. (A) The relative expression levels of ANGPTL2, CK15, and EEF1E1 proteins in cancer tissues of the control (cgn, cgpl, cghdscc, cgmdscc, and cgpdscc) and experimental groups (egn, egpl, eghdscc, egmdscc, and egpdscc) are detected by Western blot assay (n = 3). (B) Statistical analysis of the expression differences of ANGPTL2, CK15, and EEF1E1 between the experimental and control groups (n = 3). (C) Differential expression analysis of ANGPTL2, CK15, and EEF1E1 during oral mucosal carcinogenesis. (Compared between two groups, * represents statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns represents P > 0.05)

• 

  Fig. 7. Immunofluorescent staining of MAPH markers in the oral mucosal carcinogenesis. (A) The relative expression levels of ANGPTL2, CK15, and EEF1E1 proteins in the cancer tissues of the cgn, cgpl, cghdscc, cgmdscc, and cgpdscc groups were detected by immunofluorescence assay (TRITC, ×200, n = 6). (B) The relative expression levels of ANGPTL2, CK15, and EEF1E1 proteins in the cancer tissues of the egn, egpl, eghdscc, egmdscc, and egpdscc groups were detected by immunofluorescence assay (TRITC, ×200, n = 6).