Toward mitigating radiation-induced lung injury using precision cut lung slices technology (PCLS)

Abstract

Radiation induced lung injury (RILI) occurs when healthy lung tissue is exposed to radiation. Such exposure will typically occur during radiotherapy for lung or breast cancer, but may also occur as a consequence of nuclear catastrophe or radiological terrorism. Finding ways of mitigating RILI would have obvious benefits in terms of potentially allowing the development of more effective radiotherapy, as well as reducing the acute and chronic consequences of population level exposures.

Whilst previously published data indicates that prior exposure to synthetic lamellar bodies (LMS-611®), Lamellar Biomedical Ltd, Glasgow) mitigates acute RILI in an animal model, the mechanisms underlying this effect are unknown.

This research project evaluated the transcriptional changes that occur in lung tissue as a consequence of radiation exposure, and determined the effect of prior exposure to LMS-611® on these changes.

An ex vivo culture system using ovine precision cut lung slices was firstly optimised through evaluating functional indices of viability and metabolic competence, as well as imaging to determine the feasibility of delivering LMS611® to the alveolar surface of PCLS in culture. Whilst the latter aspect was qualitative in nature the imaging proved vital in indicating the persistence of low melting point agar in the alveolar air spaces, thereby informing the design of future experiments aimed at evaluating the effect of prior exposure to LMS611® on RILI. Whilst data suggested that PCLS were functionally stable over the first week of culture, questions remained as to whether such stability was accompanied by relative transcriptional quiescence – and hence would justify the notion that the PCLS reflects normal healthy lung tissue in vivo. A microarray experiment was designed and implemented to compare the time dependent change in gene expression of ovine PCLS over seven days in culture, comparing responses against a baseline of naïve unperturbed lung tissue. This study demonstrated that the transcriptional response of ovine PCLS shares significant similarity with that demonstrated for lung tissue responding to physical injury, as well as differences likely indicative of the inevitable compromises associated with ex vivo models. These observations were a crucial pivot in helping interpret the effects of LMS in mitigating RILI. In the final microarray experiment, necropsy lung segments from eight sheep were instilled with either saline or LMS-611 for 10 minutes. Thereafter the excess was poured off and each segment inflated with low melting point agar to smooth pleural confluence. After the cooled segments hardened, each was transversely sectioned into three 1cm thick slices. One slice was exposed to 6Gy radiation, one to 12Gy radiation and a third left unexposed as a control (CON), and PCLS cut from these tissues culture over a period of one week, with harvesting at time points therein to determine the transcriptional features of the radiation response, as well as the influence of LMS-611 in potentially mitigating radiation-induced effects.

The radiation response of ovine PCLS was largely consistent with that described in the literature for in vitro model systems, namely amongst downregulated genes, enrichment for biological processes relating to cell cycle, regulation of the immune system and response to external stimulus, and amongst up-regulated genes, enrichment for processes concerned with apoptosis and programmed cell death.

A large number of genes were significantly differentially regulated as a consequence of prior treatment with LMS. Notably, amongst up regulated DEGs, was noted the enrichment of terms relating to the regulation of gene transcription and translation, as well as development of the vasculature, whereas amongst down-regulated genes was evidence of enrichment of processes relating to mitochondrial function.

Weighted gene correlation network analysis identified clusters of genes sharing similar expression patterns amongst which several were enriched for genes significantly differentially regulated as a consequence of LMS pre- treatment. Amongst these one particular cluster of seven genes (LTF, LBP, SAA1, SCGB3A2, ZG16B, LPO and SPINK1) was of particular note, and promises informed insight on the mechanisms underlying the aforementioned in vivo effect of LMS pre-treatment on the acute RILI response.