News Update on tumour growth : Jan 2022

Defining the mode of tumour growth by clonal analysis

Recent studies using the isolation of a subpopulation of tumour cells followed by their transplantation into immunodeficient mice provide evidence that certain tumours1,2, including squamous skin tumours3,4,5, contain cells with high clonogenic potential that have been referred to as cancer stem cells (CSCs). Until now, CSC properties have only been investigated by transplantation assays, and their existence in unperturbed tumour growth is unproven. Here we make use of clonal analysis of squamous skin tumours using genetic lineage tracing to unravel the mode of tumour growth in vivo in its native environment. To this end, we used a genetic labelling strategy that allows individual tumour cells to be marked and traced over time at different stages of tumour progression. Surprisingly, we found that the majority of labelled tumour cells in benign papilloma have only limited proliferative potential, whereas a fraction has the capacity to persist long term, giving rise to progeny that occupy a significant part of the tumour. As well as confirming the presence of two distinct proliferative cell compartments within the papilloma, mirroring the composition, hierarchy and fate behaviour of normal tissue, quantitative analysis of clonal fate data indicates that the more persistent population has stem-cell-like characteristics and cycles twice per day, whereas the second represents a slower cycling transient population that gives rise to terminally differentiated tumour cells. Such behaviour is shown to be consistent with double-labelling experiments and detailed clonal fate characteristics. By contrast, measurements of clone size and proliferative potential in invasive squamous cell carcinoma show a different pattern of behaviour, consistent with geometric expansion of a single CSC population with limited potential for terminal differentiation. This study presents the first experimental evidence for the existence of CSCs during unperturbed solid tumour growth.[1]

Mathematical modelling of avascular-tumour growth

A system of nonlinear partial differential equations is proposed as a model for the growth of an avascular-tumour spheroid. The model assumes a continuum of cells in two states, living or dead, and, depending on the concentration of a generic nutrient, the live cells may reproduce (expanding the tumour) or die (causing contraction). These volume changes resulting from cell birth and death generate a velocity field within the spheroid. Numerical solutions of the model reveal that after a period of time the variables settle to a constant profile propagating at a fixed speed. The travelling-wave limit is formulated and analytical solutions are found for a particular case. Numerical results for more general parameters compare well with these analytical solutions. Asymptotic techniques are applied to the physically relevant case of a small death rate, revealing two phases of growth retardation from the initial exponential growth, the first of which is due to nutrient-diffusion limitations and the second to contraction during necrosis. In this limit, maximal and ‘linear’ phase growth speeds can be evaluated in terms of the model parameters.[2]

Hypoxia — a key regulatory factor in tumour growth

Cells undergo a variety of biological responses when placed in hypoxic conditions, including activation of signalling pathways that regulate proliferation, angiogenesis and death. Cancer cells have adapted these pathways, allowing tumours to survive and even grow under hypoxic conditions, and tumour hypoxia is associated with poor prognosis and resistance to radiation therapy. Many elements of the hypoxia-response pathway are therefore good candidates for therapeutic targeting.[3]

Epidermal Growth Factor Receptor Regulates Matrix Metalloproteinase-2 Activity in MDA-MB-231 Human Breast Cancer Cells

Aims and Study Design: Overexpression of epidermal growth factor receptor (EGFR) and its phosphorylated form (p-EGFR) in breast carcinomas has been associated with an increase in invasive potential and decreased survival. EGFR mediated signal transduction has been reported to be involved in regulation of matrix metalloproteinases (MMPs). Elevated MMP-2 expression and activity shows significant correlation with increased invasive potential in breast cancer. As MMP-2 plays a crucial role in tumour invasion, the role of EGFR in regulation of MMP-2 expression and activity in breast cancer was studied using the human breast adenocarcinoma cell line MDA-MB-231 as a model.

Methodology: MDA-MB-231 cells were cultured on 1 µg/ml epidermal growth factor (EGF) coated culture dishes for 24 hours. Control cells were cultured without EGF. MMP-2 activity was assayed by gelatin zymography. Expression of EGFR, MMP-2, focal adhesion kinase (FAK) and mitogen activated protein kinase (p38MAPK) and phosphorylation of EGFR were assayed by Western blot. Results: When MDA-MB- 231 cells were cultured on EGF, increased activation of MMP-2 and an overall increase in MMP-2 expression and activity was observed. The observed upregulation of MMP-2 was appreciably inhibited if cells were pre-treated with anti-EGFR antibody, thus blocking EGFR. Phosphorylation of EGFR and expression of p38MAPK and FAK were appreciably increased upon culture of cells on EGF.

Conclusion: In MDA-MB-231 breast cancer cells, EGFR-EGF interactions promote activation of MMP-2 and an increase in MMP-2 expression and activity via EGFR mediated signal transduction cascades involving FAK and p38MAPK. As elevated MMP-2 expression and activity correlate with tumour aggressiveness, tumour cell EGF interaction via EGFR might increase the invasive potential of breast cancer cells.[4]

All-Trans Retinoic Acid Downregulates Epidermal Growth Factor Receptor Mediated Regulation of Matrix Metalloproteinase-2 in B16F10 Murine Melanoma Cells

Aims and Study Design: Epidermal growth factor receptor (EGFR) regulates a number of cellular processes, including cell motility, proliferation, differentiation and survival. EGFR mediated signal transduction via mitogen activated protein kinase (MAPK) modulates expression and activity of matrix metalloproteinases (MMPs). Elevated expression and activity of MMP-2 strongly correlates with increased tumour invasiveness. As numerous studies indicate that all-trans retinoic acid (ATRA) has considerable anti-tumorigenic potential, the effect of ATRA on EGFR mediated regulation of MMP-2 via MAPK was studied in this paper using the highly metastatic murine melanoma cell line B16F10 as a model.

Methodology: B16F10 cells were cultured in the presence of ATRA (20 μM) for 6, 15 and 24 hrs. EGFR expression and phosphorylation and p38MAPK expression were assayed by Western blot. MMP-2 activity was assayed by gelatin zymography of culture supernatants. MMP-2 and TIMP-2 mRNA expression were assayed by RT-PCR. DNA fragmentation was used to assay the efficacy of ATRA in causing apoptosis.

Results: Treatment of B16F10 murine melanoma cells with ATRA (20 µM) for 24 hrs led to an appreciable downregulation of EGFR phosphorylation and expression of EGFR and p38MAPK. Treatment of B16F10 cells with ATRA also inhibited MMP-2 activity and downregulated transcription of MMP-2 while transcription of TIMP-2 was upregulated. Treatment with ATRA did not show appreciable fragmentation of DNA.

Conclusion: Downregulation of EGFR expression and phosphorylation and EGFR mediated signal transduction through p38 MAPK could lead to downregulation of MMP-2 transcription. Downregulation of MMP-2 transcription and upregulation of transcription of the MMP inhibitor TIMP-2 could result in loss of MMP-2 activity. Loss of MMP-2 activity would render cells less metastatic. Our findings indicate that treatment with ATRA can inhibit EGFR mediated regulation of MMP-2 activity in B16F10 murine melanoma cells. Such inhibition could have therapeutic potential in clinical management of tumours.[5]

Reference

[1] Driessens, G., Beck, B., Caauwe, A., Simons, B.D. and Blanpain, C., 2012. Defining the mode of tumour growth by clonal analysis. Nature, 488(7412), pp.527-530.

[2] Ward, J.P. and King, J.R., 1997. Mathematical modelling of avascular-tumour growth. Mathematical Medicine and Biology: A Journal of the IMA, 14(1), pp.39-69.

[3] Harris, A.L., 2002. Hypoxia—a key regulatory factor in tumour growth. Nature Reviews Cancer, 2(1), pp.38-47.

[4] Majumder, A. and Banerji, A., 2016. Epidermal growth factor receptor regulates matrix metalloproteinase-2 activity in MDA-MB-231 human breast cancer cells. Biotechnology Journal International, pp.1-9.

[5] Banerji, A. and Majumder, A., 2016. All-trans retinoic acid downregulates epidermal growth factor receptor mediated regulation of matrix metalloproteinase-2 in B16F10 murine melanoma cells. Annual Research & Review in Biology, pp.1-8.

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