Analysis of radiation effects in two irradiated tumor spheroid models

Abstract

Multicellular spheroids have proven suitable as three-dimensional in vivo-like models of non-vascularized micrometastases. Unlike monolayer-based models, spheroids mirror the cellular milieu and the pathophysiological gradients inside tumor nodules. However, there is limited knowledge of the radiation effects at the molecular level in spheroids of human origin. The present study is a presentation of selected cell biological processes that may easily be analyzed with methods available at routine pathology laboratories. Using gamma irradiated pancreatic neuroendocrine BON1 and colonic adenocarcinoma HCT116 spheroids as model systems, the present study assessed the radiobiological response in these models. Spheroid growth after irradiation was followed over time and molecular responses were subsequently assessed with immunohistochemistry (IHC) staining for descriptive analyses and semi-automatic grading of apoptosis, G2-phase and senescence in thin sections of the spheroids. Growth studies demonstrated the BON1 spheroids were slower growing and less sensitive to radiation compared with the HCT116 spheroids. IHC staining for G2-phase was primarily observed in the outer viable P-cell layers of the spheroids, with the 6 Gy irradiated HCT116 spheroids demonstrating a very clear increase in staining intensity compared with unirradiated spheroids. Apoptosis staining results indicated increased apoptosis with increasing radiation doses. No clear association between senescence and radiation exposure in the spheroids were observed. The present results demonstrate the feasibility of the use of multicellular spheroids of human origin in combination with IHC analyses to unravel radiobiological responses at a molecular level. The present findings inspire further investigations, including other relevant IHC-detectable molecular processes in time- and radiation dose-dependent settings.

Chemoradiotherapy screening in a novel biomimetic polymer based pancreatic cancer model

“…7,[29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48] However, most studies focus on the development of the actual 3D model and, to the best of our knowledge, there are very limited 3D studies on PDAC treatment screening (chemotherapy and radiotherapy) with most studies conducted in 3D spheroids (cell aggregates). 14,32,[34][35][36][49][50][51][52] Generally, these studies report a higher resistance of PDAC to treatment in 3D when compared to 2D cultures, a trend which is in closer alignment with in vivo studies. 34,49,52 For example, Longati et al (2013) studied the impact of the chemotherapeutic drug Gemcitabine (GEM, 0-1 mM) on the viability of PDAC spheroids and observed a higher resistance of the spheroids to the drug, as compared to a 2D culture.…”

“…14,32,[34][35][36][49][50][51][52] Generally, these studies report a higher resistance of PDAC to treatment in 3D when compared to 2D cultures, a trend which is in closer alignment with in vivo studies. 34,49,52 For example, Longati et al (2013) studied the impact of the chemotherapeutic drug Gemcitabine (GEM, 0-1 mM) on the viability of PDAC spheroids and observed a higher resistance of the spheroids to the drug, as compared to a 2D culture. The same study also reported that chemo-resistance differed amongst different cell lines with PANC-1 showing a higher resistance to GEM in comparison to BxPC-3 and Capan-1, 7 days aer drug administration.…”

“…Radiotherapy treatment on the scaffolds was performed with a clinical 250 kV X-ray irradiator, Xstrahl 300 (Xstrahl, Camberley, UK) at the Royal Surrey County Hospital NHS Foundation Trust. More specically, at the end of week 4 of culture, the scaffolds were irradiated with radiation doses of 2, 6 and 8 Gy, selected based on established in vitro experimental protocols 49,50 and in vivo regimes followed in mouse model. 55 A square eld applicator of 15 Â 15 cm was placed 3 cm above the plate surface which was placed on an epoxy resin water equivalent phantom in order to ensure a uniform radiation eld with known radiation scattering conditions.…”

“…Furthermore, a 12 days study on tumour spheroids by Al-Ramadan et al (2018), showed that spheroids of the PDAC cell line BON1 exhibited a dose dependent (0-6 Gy) increase in the apoptotic maker caspase 7 days post treatment. 49 Small animal models (mice) have also been used by different groups to study the effects of radiation on PDAC. 55,95,96 For example, Tuli et al, (2014) reported that a mouse model developed with orthotopic implant of PDAC cells, was able to survive for up to 39 days post treatment (5 Gy, single fraction radiation).…”

“…Al-Ramadan et al (2018) also used a spheroid model for assessment of radiation (0-6 Gy) on the PDAC cell line BON1 and showed a dose dependent increase in cell apoptosis within the 3D system over a period of 7 days. 49 However, even though spheroid type models are easy to fabricate and responsive to drugs, they cannot mimic robustly the TME, mainly due to their nature as well as their structural and spatial organisation. 4,20,53,54 More specically: (i) they lack mechanical stability and mechanical tunability (ii) they cannot maintain specic spatial cellular orientation (iii) simulation of mass transfer limitations which realistically occur in a dense PDAC tissue in vivo are not accurate (iv) the ECM production cannot be efficiently replicated in a spheroid structure, mainly due to the high variability of the aggregates and the nonuniform secretion of endogenous ECM (v) due to lack of porosity and perfusion, they form unrealistically high environmental gradients which do not necessarily occur in vivo, limiting their accuracy as well as the culture duration, i.e., to a few days without requiring re-suspension, however the latter would destroy the formed TME and the formed cell-matrix interactions.…”