Commentary - Journal of Cancer Immunology (2020) Volume 2, Issue 2
Protein Therapeutics from Monolayer to Spheroids- A Model for Preclinical Investigations
Neha Arora1*, Siddhartha Sankar Ghosh1,2*
1Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India
2Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India
- *Corresponding Author:
- Neha Arora
- Siddhartha Sankar Ghosh
Received date: March 27, 2020; Accepted date: May 12, 2020
Citation: Arora N, Ghosh SS. Protein Therapeutics from Monolayer to Spheroids- A Model for Preclinical Investigations. J
Cancer Immunol. 2020; 2(2): 40-43.
Copyright: © 2020 Arora N, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The arrival of recombinant insulin in the pharmaceutical market paved a new direction for the clinical potential of proteins. Thus, the pharmaceutical industry underwent a paradigm shift towards proteins as therapeutic moieties. A number of recombinant protein drugs are available and many more are in pipeline to target major diseases, such as cancer, viral diseases, cardiovascular diseases and endocrine diseases . In many cases, the recombinant protein is effective and plays an irreplaceable role in the treatment of the pathological condition, such as hemophilia, except blood coagulation factor, which has an extremely limited source, the treatment mostly relies on the recombinant coagulation factor . Many recombinant proteins are in clinical practice for more than two decades with an optimal safety and absence of side effects. As in case of growth hormone (GH) therapy, where administration of the growth hormone in patients with severe GH deficiency and hypopituitarism was found to be safe for long- and short-term usage [3,4]. In terms of cancer protein therapeutics, currently only monoclonal antibodies, targeting over-expressed receptors, are prominently available for treatment of different tumors. The development of hybridoma technology for monoclonal antibody production revolutionized the treatment regime for solid tumors. Following approval of the first monoclonal antibody, rituximab, for clinical treatment of non-Hodgkin’s lymphoma, a number of monoclonal antibodies have been generated and are at present in clinical use for treatment of solid tumors [5,6]. However, the clinical success and the long-term benefits of recombinant protein therapy for treatment of several diseases have fostered enormous interest in their application for treatment of cancer. Li and colleagues reported anti-tumor effect of novaferon, a novel recombinant protein produced by DNA shuffling of interferon-α . Evaluation of novaferon bioactivity on tumor cells displayed reduction in proliferation of HepG2
cells in vitro and in vivo. This promising pre-clinical study
has provided strong lead for clinical trial verification.
Likewise, Zhang and colleagues demonstrated HER-
2 receptor targeted activity of another recombinant
protein e23sFv-Fdt-casp6 (immune-caspasse-6) in vitro
and in vivo . The recombinant protein e23sFv-Fdtcasp6
displayed inhibition of proliferation of HER-2
over-expressing A172 and U251MG in vitro, but not U-87
MG cells with undetectable HER-2 expression. Thus,
the ability to produce targeted cytotoxicity makes the
recombinant e23sFv-Fdt-casp6 a promising therapeutic
alternative for Glioblastoma multiforme (GBM)
From the viewpoint of therapeutics, choosing the potential drug target is extremely crucial. In pathological conditions such as cancer, the major focus lies on taming the signaling pathways that have lost their inherent regulation or control, as aberrant signaling is the hallmark of this disease . One such frequently de-regulated signaling pathway associated with pathogenesis is the AKT signaling pathway [10,11]. The AKT pathway is well documented to promote growth and survival, migration, angiogenesis and transcriptional regulation. The overexpression of pro-proliferation protein AKT and the dependency of the tumor for its proliferation make the AKT signaling pathway an emerging therapeutic target [10,11]. One of the reasons for amplified AKT function is the loss or down-regulation of PTEN function. PTEN, located on chromosome 10 is a dual specificity phosphatase, catalyzing de-phosphorylation of D3 position of phosphatidylinositol 3,4,5-trisphosphate (PIP3) to generate phosphatidylinositol 4,5-diphosphate (PIP2) thereby negatively regulating the AKT signaling pathway . PTEN mutations have been reported to be a major determinant that influence tumor development
across tissues . Missense, nonsense or frameshift
mutations occur throughout the gene; however, hotspot
mutations at specific amino acids have been identified
as Arg130, Arg173 and Arg233 . Although PTEN
mutations have been associated with wide range of
cancers, uterine cancer and glioblastoma multiforme
have the highest percentages of PTEN mutations and
homozygous loss . Envisaging the crucial role of
PTEN, one of the potential therapeutic strategies is
replenishment of the PTEN function. Hence, we cloned,
purified and characterized recombinant PTEN protein
to determine its potential as an anti-proliferative
therapeutic protein .
Following selection of the potential protein drug, the next judicious step is to choose an appropriate formulation for stabilization and delivery of the protein. Proteins possess a well-defined structure that provides it a specific function, thereby not interfering with other biological processes . However, this defined structure is very delicate making it prone to degradation and disintegration. Further, some proteins function at intracellular locations making the appliance of a delivery vehicle indispensable. The field of nanomaterials has seen some remarkable advancements when it comes to assembling materials for delivery of protein, genes or drugs for therapeutic purposes, where one such notable class of nanomaterials is metallic nanoclusters [17,18]. Nanoclusters act as fluorescent probe providing an opportunity to track the moiety tagged with the nanomaterial, providing an edge over non-fluorescent class of biomaterials [19-21]. For our protein of interest PTEN, which is an intracellular protein, a delivery vehicle becomes imperative. Therefore, we formulated a novel PEGylated nanocomposite comprising of recombinant PTEN and silver nanoclusters. The silver nanoclusters synthesized using lysozyme as the template were spherical positively charged nanostructures of size 2 ± 0.5 nm. The binding of the PTEN with silver nanomaterials was followed by PEG coating to generate spherical nanocomposites of size 125 ± 10 nm. The uptake of the nanocomposites in monolayer culture of cancer cell line was determined by confocal microscopy based on the red luminescence of the nanoclusters in the nanocomposites. To assess the goal of PTEN protein therapy, we performed studies on monolayer culture of two different cancer cell lines, breast cancer MCF7 and gliobastoma U-87 MG. We demonstrated anti-proliferative and anti-invasive activity of PTEN on breast cancer cell line. In the multi-drug resistance U-87 MG we demonstrated successful combination therapy of the nanocomposites with drug erlotinib. Thus, this comprehensive study of the novel nanocomposite on cell lines provided a strong lead to the implication of PTEN in therapeutic applications . However, a successful
anti-proliferative performance in the cell culture based studies does not necessarily translate to an at par
performance in clinical trials. One obvious reason is the
lack of complexity in the monolayer culture system. This
demands evaluation of the nanocomposite or any drug at
another testing platform before having clinical relevance.
It is to be mentioned that animal models have been
routinely used in pre-clinical studies, however there is
fundamental genetic, cellular and metabolic differences
between human tumor and xenograft human tumors
. A detailed research suggests that there are notable
differences in transformation of human cells and rodent
cells. Human cells require more genetic alterations for
oncogenic transformation as compared to their murine
counterparts . This does not reduce the importance
of these studies, but suggests exploration of data from
animal studies may require careful insights. A recent
breakthrough in biological models was the upgradation
from two-dimensional cell culture (monolayer) system
to three-dimensional cell culture [25,26]. Threedimensional
(3D) culturing of cells takes into account
the spatial organization of cells, the heterogeneity and
the in vivo cell environment. With emerging research
in this field, a plethora of protocols are available to
generated spheroids, which can be scaffold free  or
scaffold dependent . Scaffold based 3D culturing
helps to recreate an environment resembling in vivo
organ structure and function thereby overcoming the
limitations of monolayer cell culture. Synthetic hydrogels
are the most widely accepted material for scaffold as they
can mimic the biological environment of extracellular
matrix . Spheroids are self-assembling cluster of
cells that represent the true depiction of morphological
changes that take place during cell transformation .
These three dimensional structures can be directly
created from a large number of cell types and provide
excellent models for studying cellular signaling, cell
migration, differentiation, survival, and growth . Having successfully achieved anti-proliferative efficacy of our recombinant PTEN protein in monolayer cultures of cancer cell lines, we decided to take the research a step forward and evaluate the potential of our construct (nanocomposite, containing PEGylated nanoclusters and recombinant PTEN) on spheroid system. We demonstrated the generation of spheroids from monolayer cultures of MCF7 and U-87 MG cell lines. A facile and reproducible forced flotation method to generate compact single spheroid in each well was employed. This scaffold-free method was found to be very simple, robust and reliable for spheroid generation. The clustering of the cells to form the 3D structure (spheroids) was easily monitored by microscopy. The objective of performing a 3D cell culture analysis was to determine the efficiency of the nanocomposite on a complex system before it can be employed for animal studies or clinical investigations. Following internalization of the nanocomposites in the spheroid, investigation of spheroid viability, cell cycle analysis and gene expression revealed successful anti proliferative activity of the nanocomposites. However, along with testing the efficacy of the nanocomposite in the complex system, the study was also performed to highlight the differences between the monolayer culture and 3D culture systems. Just like the differences in the complexity of the two systems, there were differences in their response to the treatment regime. The complexity of the spheroid model was supported by higher doses of recombinant PTEN required to achieve similar effects as established in monolayer cultures. Thus, observation of both the monolayer cell culture and 3D cell culture systems lead us to draw two major conclusions. Firstly, although the two-dimensional monolayer culture does not entirely depict the in vivo microenvironment conditions, it is still very crucial for preliminary screening of any potential therapeutic protein in order to save time, labor and cost before exploring other platforms for further testing. Secondly, the three-dimensional model provides fruitful conclusion to therapeutic screening experiments. Hence, it is anticipated that additional modifications of the spheroid protocols in future may incur 3D model a valuable platform to establish pre-clinical investigation. The essence of the article is presented in Figure 1.
Authors acknowledge financial supports of the DBT Program Support Facility (BT/PR13560/ COE/34/44/2015) and DBT NER-BPMC (BT/PR 25095/ NER/95/1011/2017). Rajib Shome, a senior research scholar at Indian Institute of Technology Guwahati is also duly acknowledged.
- Liras A. Recombinant proteins in therapeutics: haemophilia treatment as an example. International Archives of Medicine. 2008 Dec 1;1(1):4.
- Swiech K, Picanço-Castro V, Covas DT. Production of recombinant coagulation factors: Are humans the best host cells?. Bioengineered. 2017 Sep 3;8(5):462-70.
- Stochholm K, Johannsson G. Reviewing the safety of GH replacement therapy in adults. Growth Hormone & IGF Research. 2015 Aug 1;25(4):149-57.
- Gibney J, Johannsson G. Safety of growth hormone replacement therapy in adults. Expert Opinion on Drug Safety. 2004 Jul 1;3(4):305-16.
- Marcus R, Hagenbeek A. The therapeutic use of rituximab in non-Hodgkin’s lymphoma. European journal of Haematology. 2007 Jan;78:5-14.
- Weiner LM, Surana R, Wang S. Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nature Reviews Immunology. 2010 May;10(5):317-27.
- Li M, Rao C, Pei D, Wang L, Li Y, Gao K, et al. Novaferon, a novel recombinant protein produced by DNA-shuffling of IFN-α, shows antitumor effect in vitro and in vivo. Cancer Cell International. 2014 Dec 1;14(1):8.
- Zhang L, Ren J, Zhang H, Cheng G, Xu Y, Yang S, et al. HER2-targeted recombinant protein immuno-caspase-6 effectively induces apoptosis in HER2-overexpressing GBM cells in vitro and in vivo. Oncology Reports. 2016 Nov 1;36(5):2689-96.
- Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74.
- Tokunaga E, Oki E, Egashira A, Sadanaga N, Morita M, Kakeji Y, et al. Deregulation of the Akt pathway in human cancer. Current Cancer Drug Targets. 2008 Feb 1;8(1):27-36.
- Robertson GP. Functional and therapeutic significance of Akt deregulation in malignant melanoma. Cancer and Metastasis Reviews. 2005 Jun 1;24(2):273-85.
- Stambolic V, Suzuki A, De La Pompa JL, Brothers GM, Mirtsos C, Sasaki T, et al. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell. 1998 Oct 2;95(1):29-39.
- M Dillon L, W Miller T. Therapeutic targeting of cancers with loss of PTEN function. Current Drug Targets. 2014 Jan 1;15(1):65-79.
- Song MS, Salmena L, Pandolfi PP. The functions and regulation of the PTEN tumour suppressor. Nature Reviews Molecular Cell Biology. 2012 May;13(5):283-96.
- Arora N, Ghosh SS. Functional characterizations of interactive recombinant PTEN–silica nanoparticles for potential biomedical applications. RSC Advances. 2016;6(115):114944-54.
- Leader B, Baca QJ, Golan DE. Protein therapeutics: a summary and pharmacological classification. Nature reviews Drug Discovery. 2008 Jan;7(1):21-39.
- Yahia-Ammar A, Sierra D, Me?rola F, Hildebrandt N, Le Guével X. Self-assembled gold nanoclusters for bright fluorescence imaging and enhanced drug delivery. ACS Nano. 2016 Feb 23;10(2):2591-9.
- Li J, Wang W, Sun D, Chen J, Zhang PH, Zhang JR, et al. Aptamer-functionalized silver nanoclusters-mediated cell type-specific siRNA delivery and tracking. Chemical Science. 2013;4(9):3514-21.
- Yang J, Xia N, Wang X, Liu X, Xu A, Wu Z, et al. One-pot one-cluster synthesis of fluorescent and biocompatible Ag14 nanoclusters for cancer cell imaging. Nanoscale. 2015 Nov 5;7(44):18464-70..
- Das NK, Ghosh S, Priya A, Datta S, Mukherjee S. Luminescent copper nanoclusters as a specific cell-imaging probe and a selective metal ion sensor. The Journal of Physical Chemistry C. 2015 Oct 29;119(43):24657-64.
- Li J, Zhong X, Cheng F, Zhang JR, Jiang LP, Zhu JJ. One-pot synthesis of aptamer-functionalized silver nanoclusters for cell-type-specific imaging. Analytical Chemistry. 2012 May 1;84(9):4140-6.
- Arora N, Gavya S L, Ghosh SS. Multi-facet implications of PEGylated lysozyme stabilized-silver nanoclusters loaded recombinant PTEN cargo in cancer theranostics. Biotechnology and Bioengineering. 2018 May;115(5):1116-27
- Permlid AM, Roci P, Fredlund E, Fält F, Önell E, Johansson F, et al. Unique animal friendly 3D culturing of human cancer and normal cells. Toxicology in Vitro. 2019 Oct 1;60:51-60.
- Rangarajan A, Hong SJ, Gifford A, Weinberg RA. Species-and cell type-specific requirements for cellular transformation. Cancer Cell. 2004 Aug 1;6(2):171-83.
- Langhans SA. Three-dimensional in vitro cell culture models in drug discovery and drug repositioning. Frontiers in Pharmacology. 2018 Jan 23;9:6.
- Antoni D, Burckel H, Josset E, Noel G. Threedimensional cell culture: a breakthrough in vivo.International Journal of Molecular Sciences. 2015 Mar;16(3):5517-27.
- Türker E, Demirçak N, Arslan-Yildiz A. Scaffoldfree three-dimensional cell culturing using magnetic levitation. Biomaterials Science. 2018 Jun 25;6(7):1745-53.
- Cunha C, Panseri S, Villa O, Silva D, Gelain F.3D culture of adult mouse neural stem cells within functionalized self-assembling peptide scaffolds.International Journal of Nanomedicine. 2011;6:943.
- Khawar IA, Park JK, Jung ES, Lee MA, Chang S, Kuh HJ. Three dimensional mixed-cell spheroids mimic stroma-mediated chemoresistance and invasive migration in hepatocellular carcinoma. Neoplasia. 2018 Aug 1;20(8):800-12.