ESTRO 2024 - Abstract Book

S5366

Radiobiology - Tumour biology

ESTRO 2024

Introduction. Development of biological agents to combine with radiation therapy (RT) lags behind the development of anti-neoplastic drugs, which are tested with RT only at the final phases of drug development[1]. Lack of appropriate preclinical models for the early testing of RT-drug combinations may contribute to this. 3D tumour models are more physiologically relevant than 2D culture, and less expensive and time-consuming than animal surrogates[2]. Micro dissected tumour tissues (MDTs) are ex vivo 3D models derived from patients’ biopsies or xenografted tumours[3]. They preserve the integrity of the original tumour, including cancer cells and other components of the tumour microenvironment. Manipulation of MDTs in microfluidic chips (i.e., a set of micron-sized channels and wells) reduces consumption of reagents, cost and time[1]. This cancer model can be adapted to various applications including RT drug response assessment. In this project, we tested one strategy to increase the efficacy of RT through its combination with a senolytic drug, AZD4320 that inhibits antiapoptotic factors Bcl-2 and Bcl-xL[4]. RT causes treatment-induced senescence, a state in which cells are not replicating but metabolically active[5]. Treatment induced senescent cells are dependent on Bcl-2 family proteins to survive and inhibition of these pathway can be exploited to selectively target these cells[6].

Objective. The purpose of this project is to determine the toxicity of RT with and without AZD4320 senolytics on MDTs derived from tumour xenografts.

Material/Methods:

HCT116 human colorectal cancer and SK-LMS-1 human leiomyosarcoma xenografts were used to make MDTs according to an established protocol[7]. MDTs were loaded in previously developed microfluidic chips (Figure 1.A). After 2-3 days, MDTs were treated with RT ranging from 0 (CTRL) to 8Gy. HCT116 MDTs were fixed 30 minutes, 24h, and 5 days after RT. Sarcoma MDTs that received 0 or 8Gy were treated with DMSO or 0.25µM of AZD4320, 5 days after RT. Two days later, MDTs were fixed in formalin. To measure treatment response, fixed MDTs were stained for various markers such as Ki-67 (proliferation marker) and g-H2AX (DNA damage marker). The MDT chip’s media was collected at various time-points to measure circulating tumour DNA (ctDNA) using qPCR. CtNDA is cell-free DNA originated from tumour cells that are mostly released from dying cells[8]. Thus, quantifying ctNDA may represent a surrogate marker of cell death in real-time. Finally, the viability of the MDTs was measured by using a commercial kit (RealTime-Glo™ MT Cell Viability Assay). Experiments were repeated three times (N=3 tumours, n>15 MDTs per condition). One- or two-way ANOVA statistical analysis was done using GraphPad Prism (*p-value<0.05, **p value<0.01, ***p-value<0.001, ****p-value<0.0001).

Results:

HCT116 MDTs that received increasing doses of RT grew slower than control MDTs (data not shown). At the molecular level, RT reduced the nuclear area and Ki-67 expression, which is an indication of proliferation arrest and senescence (Figure 1.B and C). Moreover, 30min after RT, HCT116 MDTs demonstrated a dose response increase in the level of g H2AX foci, confirming an increasing amount of DNA damage from increasing doses of RT (Figure 1.D). By Day 5 after RT, most of DNA damage was resolved in MDTs receiving 2 and 4Gy, while more DNA damage remained in 8Gy irradiated MDTs. Persistent DNA damage, also known as DNA SCARS, is a hallmark of senescent cells.