Self-assembling peptide scaffolds as a mimic of the cancer microenvironment
Creators: name: AL BALUSHI, Noora Rasool Bakhsh
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310105
subject: Cellular interactions (incl. adhesion, matrix, cell wall)

description: The natural tumour microenvironment (TME) is complex and consists of a fibrous extracellular matrix (ECM) with multiple attachment proteins. Interactions between tumour and stromal cells and the three-dimensional (3D) architecture of the tumour are vital for tumourgenesis, progression and survival. Therefore, culturing cancer cells in 3D matrices can elicit many physiological features of the native TME by providing spatial cell–cell and cell-ECM interactions that cannot be replicated by conventional two-dimensional (2D) cell culture models. The functionalised nanofibrillar scaffolds that are made from self-assembling peptides (SAP), offer an opportunity to model the in vivo TME where cancer cells reside, proliferate,
and metastasise. Fluorenylmethyloxycarbonyl (Fmoc)-SAP hydrogels are highly customisable with a wide range of bioactive and biocompatible epitopes to mimic the native ECM cues in the TME, such as pro-adhesive epitopes to mimic integrin binding sites from ECM proteins.
Moreover, in the field of cancer treatment, SAP hydrogels have been recognised to be one of the most promising drug delivery systems for many active compounds.

This current study aimed to develop a highly versatile 3D lung tumour model in vitro by encapsulating spheroids of murine lung cancer cells (LLCs) and fibroblast cells (NOR-10) within a Fmoc-SAP hydrogel functionalised with pro-adhesive peptide sequences from laminin (IKVAV) and fibronectin (RGD) to mimic the ECM. Thus, providing the spheroids with both dimensional context and chemical support (Chapter three). In addition, this study was designed to assess the effectiveness of the Fmoc-SAP hydrogel as a drug delivery vehicle for fucoidan, anti-cancer polysaccharide, into lung tumour models in vitro (Chapter four) and primary lung tumours in vivo (Chapter five).

In Chapter three, LLC and NOR-10 mono-spheroids and co-cultured spheroids of both cell lines were grown within Fmoc-SAP hydrogels (termed as 3D), scaffold-free cultures (termed as 2.5D) or 2D cell cultures (termed as 2D). Our data indicated that culturing lung cancer spheroids in functionalised Fmoc-SAP hydrogel is effective as it significantly enhanced the overall metabolic activity of 3D co-cultured spheroids, promoted spheroids migration by reducing vinculin level and enhanced the migratory phenotype of the co-cultured spheroids by impacting the actin cytoskeleton organisation.

In Chapter four, the effectiveness of Fmoc-SAP hydrogels—as a drug delivery vehicle for fucoidan in vitro—was assessed. Fucoidan (5 mg/mL) was either co-assembled into the FmocSAP hydrogel and introduced to the embedded 3D lung tumour spheroid model established in Chapter three (termed as 3D spheroids) or resuspended in tissue culture media and delivered to lung tumour spheroids (termed as 2.5D spheroids). After 72 hours of fucoidan treatment, cell metabolic activity, apoptosis, angiogenesis and MMP2 and MMP9 activities were investigated using MTS, Bcl-2 and Bax flow cytometry, VEGF ELISA and gelatin zymography, respectively. The data revealed that fucoidan delivered by Fmoc-SAP hydrogel
to 3D lung tumour spheroids effectively decreased metabolic activity, induced apoptosis, inhibited angiogenesis and decreased the activity of MMP2 and MMP9 enzymes in 3D spheroids. No such significant effects were noted when fucoidan was delivered to 2.5D spheroids in tissue culture media. Taken together, this study may provide evidence for the effectiveness of Fmoc-SAP hydrogel as a drug delivery vehicle for fucoidan to lung tumour models in vitro.

In Chapter five, the effectiveness of Fmoc-SAP scaffolds—as a drug delivery vehicle for fucoidan in vivo—was assessed. On day five of LLC primary lung tumour induction in C57/BL6 mice, tumours were either injected with a single dose of fucoidan (5 mg/mL) coassembled in Fmoc-SAP hydrogel (noted as SAP-FCN) or fucoidan suspended in PBS (noted as FCN) or saline (noted as PBS) or Fmoc-SAP hydrogel (noted as SAP) or received no 
intratumoral injection (noted as no treatment). Tumour volume and mice survival rate were monitored on a daily basis and tumours were excised once they reached 28 days or were severely ulcerated or reached a volume greater than 1000mm3. 

After tumour excision, fixation, processing, embedding and sectioning, IHC was conducted to assess cancer cell proliferation, cancer-associated fibroblast (CAF) recruitment and angiogenesis by detecting the expression of PCNA, α-SMA and CD31 markers, respectively. TUNEL staining was conducted to detect apoptosis. The results revealed that delivering fucoidan to primary lung tumour in vivo was more efficient by Fmoc-SAP hydrogel compared to FCN. Although SAP-FCN had no effect on cancer volume, mice survival rate and angiogenesis in vivo, it significantly inhibited cancer cell proliferation, induced apoptosis and inhibited CAF recruitment compared to tumours injected with fucoidan in suspension.

In conclusion, this project presents a functional 3D lung tumour model in vitro, which we believe will be a suitable platform to investigate cancer biology and response to anti-cancer drugs in vitro. It also highlights the role of Fmoc-SAP hydrogel as an effective drug delivery vehicle for fucoidan in vitro and in vivo and can be further adopted to deliver similar tumour therapeutics in vivo.