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3D tumor spheroids: morphological alterations a yardstick to anti-cancer drug response

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Abstract

Tumor spheroids are one of the well-characterized 3D culture systems bearing close resemblance to the physiological tissue organization and complexity of avascular solid tumor stage with hypoxic core. They hold a wide-spread application in the field of pharmaceutical science and anti-cancer drug research. However, the difficulty in determining optimal technique for the generation of spheroids with uniform size and shape, evaluation of experimental outputs, or mass production often limits their usage in anti-cancer research and in high-throughput drug screening. In recent times, several studies have demonstrated various simple techniques for generating uniform-size 3D spheroids, including the hanging drop (HD), liquid overlay technique (LOT), and microfluidic approaches. Morphological alterations apart from biochemical assays, and staining techniques are suitably employed for the evaluation of experimental outcomes within 3D spheroid models. Morphological alterations in response to effective anti-cancer drug treatment in 3D tumor spheroids such as reduced spheroid size, loss of spheroid compactness and integrity or smooth surface, are highly reliable. These alterations can significantly reduce the need for biochemical assays and staining techniques, resulting in both time and cost savings. The present article specifically covers a variety of available procedures in spheroid generation. For practical applicability, we have supplemented our review study with the generation of glioblastoma U87 spheroids using HD and LOT methods. Additionally, we have also incorporated the outcome of U87 spheroid treatment with doxorubicin on spheroid morphology.

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Data availability

The data sets used and or/analyzed during the current study are available from the corresponding author upon reasonable request.

Abbreviations

2D:

Two-dimensional

3D:

Three-dimensional

5-FU:

5-Fluorouracil

5-FUChnps:

5-FU loaded chitosan nanoparticles

CAR:

Cell adhesion recognition

CPT:

Cisplatin

D:

Diameter

D2R:

Dopamine D2 receptor

DAPI:

4′,6-Diamidino-2-phenylindole dihydrochloride

DOX:

µ

FBS:

Fetal bovine serum

GBM:

Glioblastoma multiforme

GFR:

Growth factor reduced

H:

Hour

HD:

Hanging drop

IFF:

Interstitial fluid flow

LOT:

Liquid overlay technique

NA:

Not applicable

N/A:

Data not available

NCCS:

National Centre for Cell Science

OGC:

N-octanoyl glycol chitosan

PDAC:

Pancreatic ductal adenocarcinoma

PDMS:

Polydimethylsiloxane

PEGNIO:

Polyethylene glycolated niosomes

PMX:

Pemetrexed

PEGNIO:

Polyethylene glycolated niosomes

PEGNIO/D–C/t-Lyp-1:

PEGNIO/D–C conjugated with t-Lyp-1 peptide

PNIPAM-co-AA:

Poly(N-isopropylacrylamide-co-acrylic acid)

PI:

Propidium iodide

RPM:

Revolution per minute

STP:

Stiripentol

THPMs:

2-Oxo-1,2,3,4-tetrahydropyrimidines

TMZ:

Temozolomide

TPZ:

Tirapazamine

TME:

Tumor microenvironment

ULA:

Ultra low attachment

↓:

Decreased/reduced

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Acknowledgements

We are thankful to Prof. Savita Roy, Principal, Daulat Ram College, University of Delhi, for providing constant support and lab facility throughout the investigation and Late Dr Amar Jyoti for the initial efforts to conceptualize.

Funding

The study was supported by research grants from Indian Council of Medical Research (ICMR-ICRC) to ACB (No.5/13/4/ACB/ICRC/2020/NCD-III), grant from Institution of Eminence University of Delhi (Ref. No./IoE/2021/12/FRP) to ACB, grant from CSIR-UGC to A. Chhokar [573(CSIR-UGC NET JUNE 2017)]; grant from CSIR to NA (09/045(1622)/2019-EMR-I); grant from CSIR to JY (09/045(1629)/2019-EMR-I); grant from CSIR-UGC to TT (764/(CSIR-UGC NET JUNE 2019); grant from CSIR to DJ (09/0045/(11635)/2021-EMR-1) and grant from CSIR to A. Chaudhary (09/0045(12901)/2022-EMR-1). This study was partly supported by the UGC-FRPS grant [Ref. F.30–400/2017(BSR F.D.Dy.No.5326] & SERB-ECR grant (Ref.ECR/2016/000897) to late Dr Amar Jyoti, Neuropharmacology and Drug Delivery Laboratory, Daulat Ram College, University of Delhi, Delhi.

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Authors and Affiliations

  1. Molecular Oncology Laboratory, Department of Zoology, University of Delhi (North Campus), Delhi, 110007, India

    Anna Senrung, Divya Janjua, Tanya Tripathi, Nikita Aggarwal, Arun Chhokar, Joni Yadav, Apoorva Chaudhary, Udit Joshi & Alok Chandra Bharti

  2. Neuropharmacology & Drug Delivery Laboratory, Zoology Department, Daulat Ram College, University of Delhi, Delhi, 110007, India

    Anna Senrung, Sakshi Lalwani, Jasleen Kaur & Netra Ghuratia

  3. Department of Zoology, Deshbandhu College, University of Delhi, Delhi, India

    Arun Chhokar

Authors
  1. Anna Senrung
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  2. Sakshi Lalwani
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  12. Alok Chandra Bharti
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Contributions

Conceptualization: ACB and AS. Data curation: AS and ACB. Formal analysis: AS, SL, JK, NG, DJ, TT, NA, AC, JY, and AC. Funding acquisition: ACB. Investigation: AS, SL, JK, NG, DJ, and ACB. Methodology: AS, SL, JK, NG, DJ, TT, NA, AC, JY, and AC. Project administration: ACB. Resources: ACB and AS. Supervision: ACB. Validation: ACB. Visualization: AS, DJ, TT, NA, and ACB. Writing original draft: AS and ACB. Review and editing: ACB.

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Correspondence to Alok Chandra Bharti.

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Senrung, A., Lalwani, S., Janjua, D. et al. 3D tumor spheroids: morphological alterations a yardstick to anti-cancer drug response. In vitro models 2, 219–248 (2023). https://doi.org/10.1007/s44164-023-00059-8

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