Dataset Information

Tumor analysis: freeze-thawing cycle of triple-negative breast cancer cells alters tumor CD24/CD44 profiles and the percentage of tumor-infiltrating immune cells.

ABSTRACT:

Objective

The use of novel methods to characterize living tumor cells relies on well-conceived biobanks. Herein, we raised the question of whether the composition of fresh and freeze/thawed dissociated tumor samples is comparable in terms of quantitative and qualitative profiling.

Results

Breast cancer is a heterogeneous disease, encompassing luminal A and B, basal/triple-negative breast cancer (TNBC), and ERBB2-like tumors. We examined living cells dissociated from TNBC and found that a classical freeze/thaw protocol leads to a marked reduction in the number of CD45^-CD44^LowCD24^Low tumor cells. This, in turn, changed the percentage of tumor cells with certain CD44/CD24 expression patterns and changed the percentage of tumor-infiltrating immune cells. These cryopreservation-driven alterations in cellular phenotype make it impossible to compare fresh and frozen samples from the same patient directly. Moreover, the freeze/thaw process changed the transcriptomic signatures of triple-negative cancer stem cells in such a manner that hierarchical clustering no longer ranked them according to expected inter-individual differences. Overall, this study suggests that all analyses of living tumor cells should be conducted only using freshly dissociated tumors if we are to generate a robust scoring system for prognostic/predictive markers.

SUBMITTER: Le Gallo M

PROVIDER: S-EPMC6011598 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Tumor analysis: freeze-thawing cycle of triple-negative breast cancer cells alters tumor CD24/CD44 profiles and the percentage of tumor-infiltrating immune cells.

Le Gallo Matthieu M de la Motte Rouge Thibault T Poissonnier Amanda A Lavoué Vincent V Tas Patrick P Leveque Jean J Godey Florence F Legembre Patrick P

BMC research notes 20180620 1

<h4>Objective</h4>The use of novel methods to characterize living tumor cells relies on well-conceived biobanks. Herein, we raised the question of whether the composition of fresh and freeze/thawed dissociated tumor samples is comparable in terms of quantitative and qualitative profiling.<h4>Results</h4>Breast cancer is a heterogeneous disease, encompassing luminal A and B, basal/triple-negative breast cancer (TNBC), and ERBB2-like tumors. We examined living cells dissociated from TNBC and found ...[more]

PMID: 29925435

Similar Datasets

Project description:Breast cancer cells with CD44+/CD24- cell surface marker expression profile are proposed as cancer stem cells (CSCs). Normal breast epithelial cells that are CD44+/CD24- express higher levels of stem/progenitor cell associated genes. We, amongst others, have shown that cancer cells that have undergone epithelial to mesenchymal transition (EMT) display the CD44+/CD24- phenotype. However, whether all genes that induce EMT confer the CD44+/CD24- phenotype is unknown. We hypothesized that only a subset of genes associated with EMT generates CD44+/CD24- cells.MCF-10A breast epithelial cells, a subpopulation of which spontaneously acquire the CD44+/CD24- phenotype, were used to identify genes that are differentially expressed in CD44+/CD24- and CD44-/CD24+ cells. Ingenuity pathway analysis was performed to identify signaling networks that linked differentially expressed genes. Two EMT-associated genes elevated in CD44+/CD24- cells, SLUG and Gli-2, were overexpressed in the CD44-/CD24+ subpopulation of MCF-10A cells and MCF-7 cells, which are CD44-/CD24+. Flow cytometry and mammosphere assays were used to assess cell surface markers and stem cell-like properties, respectively.Two thousand thirty five genes were differentially expressed (p < 0.001, fold change >or= 2) between the CD44+/CD24- and CD44-/CD24+ subpopulations of MCF-10A. Thirty-two EMT-associated genes including SLUG, Gli-2, ZEB-1, and ZEB-2 were expressed at higher levels in CD44+/CD24- cells. These EMT-associated genes participate in signaling networks comprising TGFbeta, NF-kappaB, and human chorionic gonadotropin. Treatment with tumor necrosis factor (TNF), which induces NF-kappaB and represses E-cadherin, or overexpression of SLUG in CD44-/CD24+ MCF-10A cells, gave rise to a subpopulation of CD44+/CD24- cells. Overexpression of constitutively active p65 subunit of NF-kappaB in MCF-10A resulted in a dramatic shift to the CD44+/CD24+ phenotype. SLUG overexpression in MCF-7 cells generated CD44+/CD24+ cells with enhanced mammosphere forming ability. In contrast, Gli-2 failed to alter CD44 and CD24 expression.EMT-mediated generation of CD44+/CD24- or CD44+/CD24+ cells depends on the genes that induce or are associated with EMT. Our studies reveal a role for TNF in altering the phenotype of breast CSC. Additionally, the CD44+/CD24+ phenotype, in the context of SLUG overexpression, can be associated with breast CSC "stemness" behavior based on mammosphere forming ability.

Project description:Tumorigenic breast cancer cells characterized by CD44 expression and low or undetectable CD24 levels (CD44+/CD24-/low) may be resistant to chemotherapy and therefore responsible for cancer relapse. Paired breast cancer core biopsies before and after neoadjuvant chemotherapy or lapatinib were obtained and as single cell suspensions stained using antibodies against CD24, CD44, and lineage markers, and then analyzed by flow cytometry. Mammosphere (MS) formation in culture was compared before and after treatment. Global gene expression differences between cancer cells bearing CD44+/CD24-/low cells and all other sorted cells, and between cancer MS and the primary bulk invasive cancers were analyzed. We report that CD44+/CD24-/low tumorigenic breast cancer cells were intrinsically chemoresistant ─ chemotherapy led to increased CD44+/CD24-/low cells, increased self-renewal capacity on MS assays, and enhanced tumorigeneicity in immunocompromised SCID/Beige mice. Conversely, in patients with HER2 overexpressing tumors, the EGFR/HER2 tyrosine kinase inhibitor, lapatinib decreased CD44+/CD24-/low cells, with the majority of these patients after conventional therapy achieving pathologic complete response, a validated surrogate marker for long-term survival. Gene transcription pathways that underlie chemoresistant, MS-forming CD44+/CD24-/low cells involve genes belonging to stem cell self-renewal, Wnt signaling, and early development pathways. Experiment Overall Design: Human breast tumor samples were sorted using flow cytometry to select for cells that were CD44+ and CD24-. Gene expression profiles of these cells were compared with profiles of the other sorted cells (CD24+ and CD44-/CD24-). Experiment Overall Design: Core biopsies of primary breast tumors were taken and placed immediately in cold RPMI-1640 supplemented with 10% heat-inactivated newborn calf serum (HINCS, Invitrogen, Carlsbad, CA). Within an hour, the samples were minced and then digested in 10-15 mL of MEGM with 250-300 units/mL collagenase at 370C. The samples were filtered, washed, and then subjected to hypotonic shock to lyse red blood cells. About 106 single cells were re-suspended, incubated for 15 min at 40C with anti-CD44 (APC), anti-CD24 (FITC), and anti-lineage cocktail antibodies (PE-conjugated anti-CD2, CD3, CD10, CD16, CD18, CD31 and CD 140B) (Pharmingen, San Diego, CA) using the manufacturer’s suggested concentrations. The cells were then washed twice, re-suspended with the viability dye propidium iodide, and analyzed using Dako MoFlo flow cytometry. Side- and forward- scatter were used to eliminate debris and cell doublets, and the Lin- cells were further analyzed by CD44 and CD24 markers.

Project description:ImportanceThe association of tumor-infiltrating lymphocyte (TIL) abundance in breast cancer tissue with cancer recurrence and death in patients with early-stage triple-negative breast cancer (TNBC) who are not treated with adjuvant or neoadjuvant chemotherapy is unclear.ObjectiveTo study the association of TIL abundance in breast cancer tissue with survival among patients with early-stage TNBC who were treated with locoregional therapy but no chemotherapy.Design, setting, and participantsRetrospective pooled analysis of individual patient-level data from 13 participating centers in North America (Rochester, Minnesota; Vancouver, British Columbia, Canada), Europe (Paris, Lyon, and Villejuif, France; Amsterdam and Rotterdam, the Netherlands; Milan, Padova, and Genova, Italy; Gothenburg, Sweden), and Asia (Tokyo, Japan; Seoul, Korea), including 1966 participants diagnosed with TNBC between 1979 and 2017 (with follow-up until September 27, 2021) who received treatment with surgery with or without radiotherapy but no adjuvant or neoadjuvant chemotherapy.ExposureTIL abundance in breast tissue from resected primary tumors.Main outcomes and measuresThe primary outcome was invasive disease-free survival [iDFS]. Secondary outcomes were recurrence-free survival [RFS], survival free of distant recurrence [distant RFS, DRFS], and overall survival. Associations were assessed using a multivariable Cox model stratified by participating center.ResultsThis study included 1966 patients with TNBC (median age, 56 years [IQR, 39-71]; 55% had stage I TNBC). The median TIL level was 15% (IQR, 5%-40%). Four-hundred seventeen (21%) had a TIL level of 50% or more (median age, 41 years [IQR, 36-63]), and 1300 (66%) had a TIL level of less than 30% (median age, 59 years [IQR, 41-72]). Five-year DRFS for stage I TNBC was 94% (95% CI, 91%-96%) for patients with a TIL level of 50% or more, compared with 78% (95% CI, 75%-80%) for those with a TIL level of less than 30%; 5-year overall survival was 95% (95% CI, 92%-97%) for patients with a TIL level of 50% or more, compared with 82% (95% CI, 79%-84%) for those with a TIL level of less than 30%. At a median follow-up of 18 years, and after adjusting for age, tumor size, nodal status, histological grade, and receipt of radiotherapy, each 10% higher TIL increment was associated independently with improved iDFS (hazard ratio [HR], 0.92 [0.89-0.94]), RFS (HR, 0.90 [0.87-0.92]), DRFS (HR, 0.87 [0.84-0.90]), and overall survival (0.88 [0.85-0.91]) (likelihood ratio test, P < 10e-6).Conclusions and relevanceIn patients with early-stage TNBC who did not undergo adjuvant or neoadjuvant chemotherapy, breast cancer tissue with a higher abundance of TIL levels was associated with significantly better survival. These results suggest that breast tissue TIL abundance is a prognostic factor for patients with early-stage TNBC.

Dataset Information

Tumor analysis: freeze-thawing cycle of triple-negative breast cancer cells alters tumor CD24/CD44 profiles and the percentage of tumor-infiltrating immune cells.

Objective

Results

Publications

Tumor analysis: freeze-thawing cycle of triple-negative breast cancer cells alters tumor CD24/CD44 profiles and the percentage of tumor-infiltrating immune cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets