Journal: Frontiers in Cell and Developmental Biology

Article Title: A 3D Platform to Investigate Dynamic Cell-to-Cell Interactions Between Tumor Cells and Mesenchymal Progenitors

doi: 10.3389/fcell.2021.767253

Figure Lengend Snippet: ddPCR assays are able to simultaneously detect tumor cells and gene-modified MSCs. (A) , A 3-plex ddPCR assay was developed to quantitively evaluate tumor cells and MSCs in the VITVO50. Custom primers and probes were designed on Luc and GFP target genes to identify tumor cells and MSCs, respectively. hRPP30 was used as reference gene to estimate the total number of human cells. Due to the DNA distribution within the droplets, in the 3-plex ddPCR assay droplets were sorted out in eight different colored clusters. (B) and (C) , 4-plex ddPCR assays were set up to simultaneously investigate MSC biodistribution and antitumor effect in the ES metastatic model. gDNA was obtained from lungs and liver of mice. Because of the random distribution of DNA into the droplets, in 4-plex ddPCR assays droplets are clustered into sixteen different colored groups. The control 4-plex ddPCR assay (B) was developed combining Luc and GFP target assays with hRPP30 and mTfrc reference assays to detect human cells, TC71 Luc cells and control MSCs, localized in mice organs. The sTRAIL 4-plex ddPCR assay (C) was set up matching Luc and sTRAIL target assays with the mTfrc and hRPP30 reference assays to distinguish between TC71 Luc tumor cells and effector MSCs in mice organs.

Article Snippet: The human ES cell line TC71 (RRID: CVCL_2,213, DSMZ, Braunschweig, Germany) was cultivated in Iscove’s modified Dulbecco’s medium (IMDM, Euroclone, Milan, Italy) supplemented with 10% FBS (Carlo Erba Reagents Srl, Cornaredo, Italy), 1% L-glutamine (200 mM; BioWhittaker, Lonza, Verviers, Belgium), and 1% penicillin-streptomycin (104 UI/ml penicillin and 10 mg streptomycin/ml; Carlo Erba Reagents Srl).

Techniques: Modification, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: A 3D Platform to Investigate Dynamic Cell-to-Cell Interactions Between Tumor Cells and Mesenchymal Progenitors

doi: 10.3389/fcell.2021.767253

Figure Lengend Snippet: TC71 cell line and MSCs effectively express reporter genes. (A) , Gating strategy for the analysis of the GFP expression on MSCs transduced with GFP-encoding pCCL PGK WPRE lentiviral vector and sorted by FACS. Gate 1: forward scatter (FSC) and side scatter (SSC) area was used to enrich for intact cells (P1). Gate 2: GFP fluorescence recorded in the logarithmic scale was used to identify MSC GFP+ (P2; 93.9 ± 0.4%). (B) , Representative picture of GFP-expressing MSCs by fluorescence microscopy (original magnification ×100). (C) , Gating strategy for the analysis of the DsRed expression on TC71 Luc cells transduced with DsRed-encoding MIGR1 vector. Gate 1: FSC and SSC area was used to enrich for intact cells (P1). Gate 2: DsRed fluorescence assessed in the logarithmic scale allowed the identification of DsRed-expressing TC71 Luc cells (P2; 98.5 ± 0.7%). (D) , Representative picture of DsRed-expressing TC71 Luc cells by fluorescence microscopy (original magnification ×100).

Article Snippet: The human ES cell line TC71 (RRID: CVCL_2,213, DSMZ, Braunschweig, Germany) was cultivated in Iscove’s modified Dulbecco’s medium (IMDM, Euroclone, Milan, Italy) supplemented with 10% FBS (Carlo Erba Reagents Srl, Cornaredo, Italy), 1% L-glutamine (200 mM; BioWhittaker, Lonza, Verviers, Belgium), and 1% penicillin-streptomycin (104 UI/ml penicillin and 10 mg streptomycin/ml; Carlo Erba Reagents Srl).

Techniques: Expressing, Transduction, Plasmid Preparation, Fluorescence, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: A 3D Platform to Investigate Dynamic Cell-to-Cell Interactions Between Tumor Cells and Mesenchymal Progenitors

doi: 10.3389/fcell.2021.767253

Figure Lengend Snippet: The VITVO50-based fluidic circuit allows to investigate dynamic cell-to-cell interactions between tumor nodules and MSCs. (A) , Scan of nine representative fields of the VITVO50 matrix colonized by TC71 Luc cells (red; Objective 4×). (B) , Representative picture taken at a higher magnification showing the formation of in vivo-like nodules of tumor cells in the VITVO50 matrix (red cell aggregates; Objective 10×) (C) Mean (SD) number of tumor cells colonizing VITVO50 bioreactor derived from escape data. Numbers of independent experiments n = 3. (D) , Bioluminescent signal after luciferin administration was used to quantify the viability of TC71 Luc cells seeded on the matrix. Data are expressed as the mean (SD). Numbers of independent experiments n = 3. (E) , Scan of nine representative fields of the VITVO50 matrix to evaluate interaction between TC71 Luc cells (red) and MSCs (green; Objective 4×). (F) , Representative picture taken at a higher magnification showing MSCs trapped between the fibers of VITVO50 matrix and interacting with tumor nodules (Objective 10×). (G) , Mean (SD) of the percentage of tumor cells (red) and MSCs (green) within the VITVO50 matrix obtained using the 3-plex ddPCR assay. Numbers of independent experiments n = 4. (H) , Ratio between the number of MSCs and tumor cells on VITVO50 matrix expressed as mean (SD). Numbers of independent experiments n = 4.

Article Snippet: The human ES cell line TC71 (RRID: CVCL_2,213, DSMZ, Braunschweig, Germany) was cultivated in Iscove’s modified Dulbecco’s medium (IMDM, Euroclone, Milan, Italy) supplemented with 10% FBS (Carlo Erba Reagents Srl, Cornaredo, Italy), 1% L-glutamine (200 mM; BioWhittaker, Lonza, Verviers, Belgium), and 1% penicillin-streptomycin (104 UI/ml penicillin and 10 mg streptomycin/ml; Carlo Erba Reagents Srl).

Techniques: In Vivo, Derivative Assay