Uncertainties Associated with Clonogenic Assays using a Cs-137 Irradiator and Ir-192 Afterloader: A Comprehensive Compilation for Radiation Researchers.

Clonogenic assays are the gold standard for measuring cell clonogenic survival and enable quantification of a cell line's radiosensitivity through the calculation of the surviving fraction, the ratio of cell clusters (colonies) formed after radiation exposure compared to the number formed without exposure. Such studies regularly utilize Cs-137 irradiators. While uncertainties for specific procedural aspects have been described previously, a comprehensive review has not been completed. We therefore quantified uncertainties associated with clonogenic assays performed using a Cs-137 Shepherd irradiator, and a recently established brachytherapy afterloader in vitro radiation delivery apparatus (BAIRDA), through a series of experiments and a literature review. The clonogenic assay is subject to uncertainties that affect the determination of the surviving fraction (e.g., accuracy of the number of cells seeded, potential effects of hypothermia, and the threshold number of cells for a cluster to be identified as a colony). Furthermore, dose delivery uncertainties related to both the Cs-137 irradiator and BAIRDA were also quantified. The combined standard (k = 1) uncertainty was ± 6.0% in the surviving fraction for the Cs-137 irradiator (±6.3% for BAIRDA), up to ± 2.2% in the dose delivered by the Cs-137 irradiator, and up to ± 4.3% in the dose delivered by BAIRDA. The largest individual uncertainties were associated with the number of cells seeded on a plate (3.4%) and inter-observer variability in counting (4.1%), suggesting that effective reduction of uncertainties in the conduct of the clonogenic assay may provide the greatest relief on the uncertainty budget. Finally, measurable impact on experimental findings was assessed by applying this uncertainty to clonogenic assays of SW756 cells using either a Cs-137 irradiator or BAIRDA, introducing a maximum shift in the reported radiobiological parameters α/ß and T1/2 of 0.3 Gy and 0.4 h, respectively, while the 95% confidence interval increased by 0.5 Gy and decreased by 0.4 h, respectively. Though the overall impact on radiobiological parameter estimation was small, the individual uncertainties could have a significant influence in other applications of in vitro experiments in radiation biology. Hence, better understanding of the uncertainties associated with both clonogenic assays and the radiation source used can improve the accuracy of experimental analysis and reproducibility of the results.

BAIRDA: a novelin vitrosetup to quantify radiobiological parameters for cervical cancer brachytherapy dose estimations.

Objective. Brachytherapy (BT) dose prescriptions for locally advanced cervical cancer are made with account for the radiobiological parameters,α/ßratio and halftime of repair (T1/2). However, a wide range of parameter values has been reported which can challenge commonly held equivalencies between dose prescriptions. This is the first reported study that aims to develop anin vitroexperimental technique using clinical high-dose-rate (HDR) and pulsed-dose-rate (PDR) Ir-192 brachytherapy afterloaders to quantify these parametersin vitroand to contextualize findings within contemporary practice.Approach. To efficiently quantifyα/ßandT1/2,in vitroexperiments more reflective of clinical BT practice than traditional clonogenic survival assays were developed and applied to four squamous cell carcinoma cell lines (CaSki, C-33A, SiHa, and SW756). Radiation was delivered using single acute and fractionated dose treatments with a conventional irradiator and clinical HDR and PDR BT afterloaders. For the latter, a novelbrachytherapyafterloaderin vitroradiationdeliveryapparatus (BAIRDA) was developed.Main Results. Theα/ßandT1/2values determined using BAIRDA and the conventional irradiator showed close agreement, validating the novel apparatus and technique. For CaSki, C-33A, SiHa, and SW756, the BAIRDA-measuredα/ßratios (5.2 [4.6-5.8], 5.6 [4.5-6.6], 6.3 [4.9-7.7], and 5.3 [4.7-6.0] Gy, respectively) were consistently smaller, while theT1/2(3.3 [2.7-3.9], 2.7 [2.0-3.3], 2.8 (2.4-3.1], and 4.8 [4.1-5.4] hours) larger, than the widely accepted values in clinical practice (α/ß= 10 Gy;T1/2 = 1.5 h).Significance.In vitroexperiments using BAIRDA provided evidence for differences between the conventionally selected and experimentally determinedα/ßratio andT1/2. Treatment regimens using HDR-BT and PDR-BT, designed to deliver equivalent radiobiological doses based on conventional values, were shown to differ by up to 27 Gy EQD2 - an effect that could impact treatment outcomes in cervical cancer. Furthermore, with BAIRDA, we have developed a novel method for radiobiological research in BT.

Range of SHH signaling in adrenal gland is limited by membrane contact to cells with primary cilia.

The signaling protein Sonic Hedgehog (SHH) is crucial for the development and function of many vertebrate tissues. It remains largely unclear, however, what defines the range and specificity of pathway activation. The adrenal gland represents a useful model to address this question, where the SHH pathway is activated in a very specific subset of cells lying near the SHH-producing cells, even though there is an abundance of lipoproteins that would allow SHH to travel and signal long-range. We determine that, whereas adrenal cells can secrete SHH on lipoproteins, this form of SHH is inactive due to the presence of cosecreted inhibitors, potentially explaining the absence of long-range signaling. Instead, we find that SHH-producing cells signal at short range via membrane-bound SHH, only to receiving cells with primary cilia. Finally, our data from NCI-H295R adrenocortical carcinoma cells suggest that adrenocortical tumors may evade these regulatory control mechanisms by acquiring the ability to activate SHH target genes in response to TGF-ß.