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Molecular heterogeneity in breast carcinoma cells with increased invasive capacities

Giulia Negro
Giulia Negro
, 
Bertram Aschenbrenner
Bertram Aschenbrenner
, 
Simona Kranjc Brezar
Simona Kranjc Brezar
, 
Maja Cemazar
Maja Cemazar
, 
Andrej Coer
Andrej Coer
, 
Gorana Gasljevic
Gorana Gasljevic
, 
Dragana Savic
Dragana Savic
, 
Maxim Sorokin
Maxim Sorokin
, 
Anton Buzdin
Anton Buzdin
, 
Maurizio Callari
Maurizio Callari
, 
Irma Kvitsaridze
Irma Kvitsaridze
, 
Anahid Jewett
Anahid Jewett
, 
Mariela Vasileva-Slaveva
Mariela Vasileva-Slaveva
, 
Ute Ganswindt
Ute Ganswindt
, 
Ira Skvortsova
Ira Skvortsova
 e 
Sergej Skvortsov
Sergej Skvortsov
   | 14 feb 2020
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Article Category: Research Article
Pubblicato online: 14 feb 2020
Pagine: 103 - 118
Ricevuto: 10 gen 2020
Accettato: 30 gen 2020
DOI: https://doi.org/10.2478/raon-2020-0007
© 2020 Giulia Negro, Bertram Aschenbrenner, Simona Kranjc Brezar, Maja Cemazar, Andrej Coer, Gorana Gasljevic, Dragana Savic, Maxim Sorokin, Anton Buzdin, Maurizio Callari, Irma Kvitsaridze, Anahid Jewett, Mariela Vasileva-Slaveva, Ute Ganswindt, Ira Skvortsova, Sergej Skvortsov, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

Holographic microscopy of breast cancer cells. Parental and invasive MDA-MB-231, T47D, and Au565 cells were analysed for their morphology using 3D tomographic microscope as described in Materials and Methods. Arrow 1: filopodia; Arrow 2: mitochondria; Arrow 3: lipid droplets.
Holographic microscopy of breast cancer cells. Parental and invasive MDA-MB-231, T47D, and Au565 cells were analysed for their morphology using 3D tomographic microscope as described in Materials and Methods. Arrow 1: filopodia; Arrow 2: mitochondria; Arrow 3: lipid droplets.

Figure 2

Breast cancer cell migration and invasion. (A) Differences in migration of parental and INV breast carcinoma cells. (B) Invasion of parental and invasive breast carcinoma cells through the collagen- and laminin coated membranes. Columns represent the mean value including standard deviation obtained from three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001).
Breast cancer cell migration and invasion. (A) Differences in migration of parental and INV breast carcinoma cells. (B) Invasion of parental and invasive breast carcinoma cells through the collagen- and laminin coated membranes. Columns represent the mean value including standard deviation obtained from three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 3

Expression of CD44+/CD24- cancer stem cell markers in breast carcinoma cells. Columns represent the mean value including standard deviation obtained from three independent experiments (**p < 0.01; ***p < 0.001).
Expression of CD44+/CD24- cancer stem cell markers in breast carcinoma cells. Columns represent the mean value including standard deviation obtained from three independent experiments (**p < 0.01; ***p < 0.001).

Figure 4

ALDH1 expression and activity in breast cancer cells. (A) Exponentially growing parental and INV breast carcinoma cells were collected for Western blot analysis. Total protein extracts were prepared from the cells and then processed for immunoblotting using antibodies to detect ALDH1A3. GAPDH was used as a loading control. IDV was determined as described in the section Materials and Methods. (B) ALDH1 activities were determined in the investigated parental and INV cells. Columns represent the mean value including standard deviation obtained from three independent experiments (**p < 0.01; ***p < 0.001).
ALDH1 expression and activity in breast cancer cells. (A) Exponentially growing parental and INV breast carcinoma cells were collected for Western blot analysis. Total protein extracts were prepared from the cells and then processed for immunoblotting using antibodies to detect ALDH1A3. GAPDH was used as a loading control. IDV was determined as described in the section Materials and Methods. (B) ALDH1 activities were determined in the investigated parental and INV cells. Columns represent the mean value including standard deviation obtained from three independent experiments (**p < 0.01; ***p < 0.001).

Figure 5

Cell proliferation rate in breast carcinoma cells. Parental and INV cells were evaluated for their doubling time as described in the Materials and Methods. For statistical evaluation, mean values and SD were calculated using at least three independent experiments; significance was determined by paired Student’s t-test (*p < 0.05; **p < 0.01).
Cell proliferation rate in breast carcinoma cells. Parental and INV cells were evaluated for their doubling time as described in the Materials and Methods. For statistical evaluation, mean values and SD were calculated using at least three independent experiments; significance was determined by paired Student’s t-test (*p < 0.05; **p < 0.01).

Figure 6

Bioinformatic analysis of proteins and pathways dysregulated in the INV compared to parental breast carcinoma cells. Overlap of up-regulated (A, B) or down-regulated (C, D) genes (A, C) and molecular pathways (B, D) between the investigated INV breast carcinoma cells. Top 10% features (proteins or pathways) were selected for the analysis. (E, F) Distribution of overlapped proteins for randomly selected groups (1000 random permutations).
Bioinformatic analysis of proteins and pathways dysregulated in the INV compared to parental breast carcinoma cells. Overlap of up-regulated (A, B) or down-regulated (C, D) genes (A, C) and molecular pathways (B, D) between the investigated INV breast carcinoma cells. Top 10% features (proteins or pathways) were selected for the analysis. (E, F) Distribution of overlapped proteins for randomly selected groups (1000 random permutations).

Figure 7

Visualization of pathways involved in breast cancer cell invasiveness. (A) “NCI Syndecan 3 mediated signaling events Main Pathway” (B) “PECAM1_interactions_Main_Pathway”; (C) “reactome Regulation of innate immune responses to cytosolic DNA Main Pathway”; (D) “ILK Signaling Pathway Actin Polymerization Cytoskeletal Reorganization”. The pathway is shown as an interacting network, where green arrows indicate activation, red arrows indicate inhibition. Color depth of each node of the network corresponds to the logarithms of the case-to-normal (CNR) expression rate for each node, where “normal” is expression level in the control group, the scale represents extent of up/downregulation.
Visualization of pathways involved in breast cancer cell invasiveness. (A) “NCI Syndecan 3 mediated signaling events Main Pathway” (B) “PECAM1_interactions_Main_Pathway”; (C) “reactome Regulation of innate immune responses to cytosolic DNA Main Pathway”; (D) “ILK Signaling Pathway Actin Polymerization Cytoskeletal Reorganization”. The pathway is shown as an interacting network, where green arrows indicate activation, red arrows indicate inhibition. Color depth of each node of the network corresponds to the logarithms of the case-to-normal (CNR) expression rate for each node, where “normal” is expression level in the control group, the scale represents extent of up/downregulation.

Figure 8

Tumorigenic abilities of parental and INV breast cancer cells. Tumor growth curves of breast cancer xenografts, parental (MDA-MB-231, T47D, Au565, ) and INV counterparts (MDA-MB-231-INV, T47D-INV, Au565-INV) are received as descibed in the section Materials and Methods.
Tumorigenic abilities of parental and INV breast cancer cells. Tumor growth curves of breast cancer xenografts, parental (MDA-MB-231, T47D, Au565, ) and INV counterparts (MDA-MB-231-INV, T47D-INV, Au565-INV) are received as descibed in the section Materials and Methods.

Figure 9

Representative histological images of breast cancer xenografts. Tumors originating from parental (A) and invasive (B) MDA-MB-231, T47D and Au565 breast carcinoma cells were stained with H&E for evaluation of tumor morphology (200× magnification). Collagen (blue) was stained using Masson`s staining (200× magnification). Ki-67 proliferative tumor cells are stained brown (600× magnification).
Representative histological images of breast cancer xenografts. Tumors originating from parental (A) and invasive (B) MDA-MB-231, T47D and Au565 breast carcinoma cells were stained with H&E for evaluation of tumor morphology (200× magnification). Collagen (blue) was stained using Masson`s staining (200× magnification). Ki-67 proliferative tumor cells are stained brown (600× magnification).

Figure 10

Histological analysis of parental and INV breast xenografts. Tumors originated from the parental (MDA-MB-231, T47D, Au565) and INV breast carcinoma cells (MDA-MB-231-INV, T47D-INV, Au565-INV) were evalueated for desmoplasia (intratumoral collagen content) and percentage of proliferative cells (Ki67 expression) (*p < 0.05; **p < 0.01; ***p < 0.001).
Histological analysis of parental and INV breast xenografts. Tumors originated from the parental (MDA-MB-231, T47D, Au565) and INV breast carcinoma cells (MDA-MB-231-INV, T47D-INV, Au565-INV) were evalueated for desmoplasia (intratumoral collagen content) and percentage of proliferative cells (Ki67 expression) (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 11

Lymph nodes of mice bearing breast cancer xenografts originated from parental (MDA-MB-231, T47D, Au565) and INV counterparts (Au565-INV, MDA-MB-231-INV, T47D-INV). (A) Representative images of macromorphology of the lymph nodes; (B) Cumulative weight of lymph nodes (AM+SE of 6 mice per group).
Lymph nodes of mice bearing breast cancer xenografts originated from parental (MDA-MB-231, T47D, Au565) and INV counterparts (Au565-INV, MDA-MB-231-INV, T47D-INV). (A) Representative images of macromorphology of the lymph nodes; (B) Cumulative weight of lymph nodes (AM+SE of 6 mice per group).

Figure 12

Histology of lymph nodes of mice bearing breast cancer xenografts. (A) HE staining in lymph nodes corresponding to the parental MDA-MB-231, T47D, Au565 and (B) invasive MDA-MB-231-INV, T47D-INV, Au565-INV xenografts. Representative images of lymph nodes taken under 400 × magnification. Brown staining represent pan-cytokeratin positive tumor cells in lymph node.
Histology of lymph nodes of mice bearing breast cancer xenografts. (A) HE staining in lymph nodes corresponding to the parental MDA-MB-231, T47D, Au565 and (B) invasive MDA-MB-231-INV, T47D-INV, Au565-INV xenografts. Representative images of lymph nodes taken under 400 × magnification. Brown staining represent pan-cytokeratin positive tumor cells in lymph node.

Proliferation abilities and collagen content in breast tumor xenografts

Tumor cell line% of proliferating% of collagen
cells (mean ±se)(mean ±se)
MDA-MB-23146.1 ± 3.118.7 ± 2.0
MDA-MB-231-INV69.8 ± 4.7*22.9 ± 3.5
T47D43.3 ± 2.33.3 ± 0.7
T47D-INV22.9 ± 2.4***21.6 ±1 .9***
Au56524.3 ± 1.732.3 ± 1.8
Au565-INV57.7 ± 3.5***6.1 ± 1.4***
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