Tumour Microenvironment-Mediated Drug Resistance in Neuroblastoma

Pediatrics Oncology Life Sciences Immunology Medicine Cell Biology Biochemistry
Neuroblastoma Drug Resistance Tumour Microenvironment TGF-β1 Nuclear Factor-κB Therapy

Background

Research Background:

Both intrinsic and extrinsic factors in the tumor microenvironment (TME) can lead to treatment resistance. This paper reveals that the Transforming Growth Factor (TGF)-β1 produced within the TME increases drug resistance in neuroblastoma (NB) cells. Drug resistance is the main cause of cancer treatment failure, usually due to intrinsic genomic and chromosomal instability in cancer cells, as well as epigenetic changes associated with selective pressure and tumor heterogeneity. Additionally, drug resistance may also be caused by external factors, such as abnormal blood flow and the impact of TME, known as Environment-Mediated Drug Resistance (EMDR).

Neuroblastoma is the most common extracranial solid tumor in children and has a high risk of recurrence. About 50% of NB children are classified as high-risk cancer. Despite the use of comprehensive treatments, including surgery, myeloablative chemotherapy, radiotherapy, and immunotherapy, more than 50% of children still die of refractory or recurrent disease.

Research Origin

The authors of this paper include Kévin Louault, Laurence Blavier, Men-Hua Lee, Rebekah J. Kennedy, G. Esteban Fernandez, Bruce R. Pawel, Shahab Asgharzadeh, and Yves A. Declerck. They belong to the Cancer and Blood Disease Research Center at Children’s Hospital Los Angeles and the University of Southern California, The Saban Research Institute, Pathology and Laboratory Medicine, and the Department of Biochemistry and Molecular Medicine. The paper was published in the British Journal of Cancer, with an online publication date of May 28, 2024.

Detailed Research Process

  1. Preparation of Cell Lines and Human Mononuclear Cells:

    • The NB cell lines used included CHLA-255, SK-N-AS, SK-N-SH, CHLA-136, SK-N-BE(2), including both MYCN and C-MYC expressed and unmodified. NB cell lines were obtained and cultured from Children’s Hospital Los Angeles.
    • MSC was obtained and cultured from the bone marrow of healthy donors. MN was isolated from the peripheral blood of healthy blood donors and used in experiments.

  2. In Vitro Co-Culture Experiments:

    • Co-culture experiments were conducted using Transwell. The dual co-culture NB:MN ratio was 1:1, and the NB:MSC ratio was 4:1; the triple co-culture ratio was 4:4:1 (NB:MN:MSC).
    • Different cell components were cultured separately, treated with different concentrations of drugs or human recombinant TGFB1, with experiment time spans ranging from 10 minutes to 3 days.

  3. Drug Sensitivity Tests:

    • NB cell sensitivity to Doxorubicin (Dox) and Etoposide (Etop) was tested under various cell co-culture settings, using the trypan blue exclusion method to measure viable cells.

  4. Protein Detection and Analysis:

    • Western Blot was used to analyze the expression of various proteins in NB cell lysates.
    • Co-Immunoprecipitation (Co-IP) experiments were performed to identify protein complexes.
    • Nuclear extraction experiments were conducted to assess the separation and expression of nuclear and cytoplasmic proteins.

  5. Gene Knockout Experiments:

    • Gene knockout experiments were conducted using RNA interference (RNAi) methods to detect the impact of gene knockout on target protein expression and cell drug resistance.

  6. Immunohistochemistry and Animal Model Experiments:

    • Chemotherapy drug sensitivity was tested in NB tumor transplanted models, using immunodeficient mice for in vivo drug response experiments.
    • Immunohistochemical analysis was used to detect the expression of p-NF-κB in different NB tumor tissues.

Detailed Research Results

  1. TGF-β1-Induced EMDR:

    • Results indicated that after co-culturing with TME cells for 72 hours, NB cells had a significantly increased IC50 for Dox and Etop compared to NB cells cultured alone. Western Blot analysis showed that TME cells could increase the expression of multi-drug resistance proteins MRP1 and MDR1, as well as survival proteins Bcl-XL, Mcl-1, and Survivin.
    • Using the TGF-βR1/ALK5 inhibitor Galunisertib significantly restored the drug sensitivity of NB cells, confirming the role of TGF-β1 in EMDR.

  2. TGF-β1-Activated NF-κB:

    • In NB cells, TGF-β1 not only induced the phosphorylation of Smad2 but also the phosphorylation and nuclear translocation of NF-κB p65.
    • Using the NF-κB nuclear translocation inhibitor JSH-23 inhibited the upregulation of multi-drug resistance proteins and survival proteins in TGF-β1-treated NB cells, confirming the crucial role of NF-κB in TGF-β1-induced drug