Here, we detail a straightforward live imaging approach for quantifying the sensitivity of patient-derived tumor organoids to ionizing radiation.
Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not always) because of differences in the ability of malignant cells to repair oxidative DNA damage as elicited by ionizing rays. Clonogenic assays have been employed for decades to assess the sensitivity of cultured cancer cells to ionizing irradiation, largely because irradiated cancer cells often die in a delayed manner that is difficult to quantify with short-term flow cytometry- or microscopy-assisted techniques. Unfortunately, clonogenic assays cannot be employed as such for more complex tumor models, such as patient-derived tumor organoids (PDTOs). Indeed, irradiating established PDTOs may not necessarily abrogate their growth as multicellular units, unless their stem-like compartment is completely eradicated. Moreover, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the sensitivity of malignant cells to RT in the context of established PDTOs. Here, we detail an adaptation of conventional clonogenic assays that involves exposure of established PDTOs to ionizing radiation, followed by single-cell dissociation, replating in suitable culture conditions and live imaging. Non-irradiated (control) PDTO-derived stem-like cells reform growing PDTOs with a PDTO-specific efficiency, which is negatively influenced by irradiation in a dose-dependent manner. In these conditions, PDTO-forming efficiency and growth rate can be quantified as a measure of radiosensitivity on time-lapse images collected until control PDTOs achieve a predefined space occupancy.
External beam radiation therapy (RT) is one of the mainstays of modern oncology, reflecting not only a pronounced anticancer activity associated with a well-defined spectrum of generally manageable side effects1, but also an exceptionally widespread clinical availability (most cancer centers in developed countries are equipped with modern linear accelerators for external beam RT)2. In line with this notion, RT is globally employed with success for both curative purposes, generally in the context of early-stage disease3,4, and palliative applications, to contain symptoms (e.g., pain) from metastatic tumors5. That said, not all malignancies are equally sensitive to RT, reflecting a number of cancer cell-intrinsic and microenvironmental features6,7,8,9. As a standalone example, various cancer-associated genetic and epigenetic alterations influencing DNA repair and redox homeostasis have been associated with increased or decreased intrinsic radiosensitivity, largely reflecting the ability of RT to kill cancer cells by causing direct and reactive oxygen species (ROS)-dependent damage to DNA and other macromolecules10,11,12,13.
Over the past decades, clonogenic assays have been routinely employed to assess the radiosensitivity of cultured human and murine cancer cells14,15,16. Indeed, while chemotherapeutics and other stressors often kill malignant cells in a fairly rapid manner, RT employed at clinically relevant doses most often elicit a delayed form of cell death that cannot be simply quantified with short-term flow cytometry- or microscopy-assisted techniques, such as measuring the cellular uptake of vital dyes (which selectively stain dead cells) 24-72 h after exposure to a cytotoxic stimulus17. Besides being straightforward and relying on rather inexpensive reagents, clonogenic assays have been particularly convenient as they allow for the implementation of the so-called "linear quadratic" model, which is a fairly simple mathematical tool for the quantitative analysis of RT dose response18,19. Along similar lines, cultured cancer cells (including established cell lines as well as primary, patient-derived cells) have provided a convenient platform for testing various aspects of cancer biology, including (but not limited to) sensitivity to treatment in a rather straightforward but simplified manner, one major limitation being the absence of a structured tumor microenvironment (TME)20,21. Indeed, two-dimensional cancer cell cultures are intrinsically unable to recapitulate cell-cell communications and interactions with the extracellular matrix and as they occur in cancer22,23. Patient-derived tumor organoids (PDTOs) have emerged as tumor models that at least in part circumvent such limitation24,25,26.
Unfortunately, conventional clonogenic assays cannot be employed to assess the radiosensitivity of PDTOs. On the one hand, irradiating established PDTOs may not necessarily compromise their growth as a multicellular unit, unless their stem-like compartment – which is responsible for the formation and maturation of PDTOs but exhibits increased resistance to DNA damage as compared to more differentiated PDTO-composing cells27– is completely eradicated. On the other hand, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the radiosensitivity of PDTO-composing (including stem-like) cells as exhibited in the context of formed PDTOs. Here, we detail a technique that harnesses live imaging of breast cancer PDTOs to monitor the PDTO-forming efficacy and growth rate of PDTO-derived cells previously exposed to ionizing irradiation compared to their unirradiated counterparts. With required variations largely reflecting the differential biology of individual PDTOs (e.g., culture media requirements, growth rate), we expect this protocol to be suitable for the study of radiosensitivity in a wide panel of PTDOs of both mammary and non-mammary origin.
The reagents and equipment used in the study are listed in the Table of Materials.
1. Organoid culture
NOTE: TNBC#1 PDTOs were established in our lab based on tumor tissue surgically removed from a patient with triple-negative breast cancer (TNBC) who provided informed consent to participate in a biobanking protocol (IRB21-06023682). After validation by histology and RNA sequencing (RNAseq), TNBC#1 PDTOs are cultured in 66% matrigel drops (so-called 'domes') in enriched DMEM/F12 culture medium (Table 1) and passaged every 2-3 weeks at a 1:2-1:10 ratio28.
2. Radiation
3. Dissociation
4. Live imaging
5. Analysis
TNBC#1 PDTOs were exposed to a single radiation dose of 0 (unirradiated controls), 2 Gy, 4 Gy, 6 Gy, 8 Gy, or 10 Gy on day 0. Immediately thereafter, PDTOs were dissociated to obtain a single-cell suspension for each experimental condition. PDTO-derived cells were next seeded in 48-well plates within 66% matrigel domes (50 µL each) deposited at the center of the wells, in 3 technical replicates per condition. Plates were placed in a live imaging system and imaged every 6 h using the organoid module 4X objective for a total of 30 days. Irradiation of TNBC#1 PDTOs with RT in a single dose of 2 Gy significantly delayed PDTO regrowth, while radiation doses ≥4 Gy (up to 10 Gy) completed prevented the regeneration of growing PDTOs (Figure 1).
Figure 1: Live imaging of patient-derived tumor organoids (PDTOs). Breast cancer TNBC#1 patient-derived tumor organoids (PDTOs) were left untreated or exposed to the indicated radiation dose, shortly followed by dissociation, replating, and imaging with a live cell imaging system. Representative images upon contrast adjustment (A) and quantitative assessments (B, mean ± SEM) are reported. Image scale: 1.19 x 1.16 mm (1.38 mm2). Scale bar: 400 µm; insets: 200 µm. ****p < 0.001 (two-way ANOVA, as compared to 0 Gy); ####p < 0.001 (two-way ANOVA, as compared to 2 Gy). Please click here to view a larger version of this figure.
Component | Concentration |
DMEM F/12 | 1X |
GlutaMax | 1X |
Hepes | 10 mM |
PenStrep | 100 U/mL |
B27 | 1X |
nAc | 1.25 mM |
Nicotinamide | 10 mM |
TGFbeta Receptor Inhibitor A83-01 | 0.5 μM |
p38 MAP inhibitor p38i SB202190 | 1 μM |
Noggin | 10% |
Rspondin Media | 10% |
FGF10 | 20 ng/mL |
FGF7 | 5 ng/mL |
NR (Heregulin) | 5 nM |
Primocin | 100 μg/mL |
Y-27632 (RhoKi) | 5 μM |
Epidermal Growth Factor hEGF | 5 ng/mL |
Table 1: PDTO culture medium composition.
Here, we describe an adaptation of conventional clonogenic assays that harnesses breast cancer PDTOs and live imaging to quantify PDTO radiosensitivity based on (1) the persistence of PTDO-forming stem-like cells upon PDTO irradiation in vitro, and (2) the growth rate of the PDTOs these cells (may) generate. Critical steps of this protocol include (1) the establishment of PDTOs to a dome occupancy enabling good viability, (2) PDTO exposure to ionizing irradiation at different doses, including mock irradiated control conditions; (3) PDTO dissociation into single cells; (4) replating of PDTO-derived single cells; and (5) live imaging until a predefined dome occupancy in control conditions.
With minimal modifications, mostly linked to PDTO-forming efficiency and PDTO growth rate in control conditions (which are likely to vary considerably across different PDTOs), we expect this protocol to be adaptable to multiple commercially available live imaging platforms, as well as to numerous PDTOs, of both mammary and non-mammary derivation. On the one hand, baseline PDTO-forming efficiency upon single-cell dispersion is expected to influence the number of single cells to be reseeded to obtain new PDTOs at a density that enables not only image-based PDTO quantification but also the normal growth of untreated PDTOs. On the other hand, the PDTO growth rate is expected to affect the duration of the assay, as the merging of adjacent PDTOs should be avoided to preserve optimal analytical capacities. Thus, determining PDTO-forming efficiency and PDTO growth rate in control conditions for each individual PDTO is critical for the correct implementation of this protocol.
While this method is found to be fairly straightforward, it has some limitations. First, an excessively low number of PDTO-forming cells may impair the regeneration of normally growing PDTOs upon single-cell dissociation. Indeed, while routine PDTO passaging can be achieved by incomplete dissociation, quantitative assessments, as offered by this protocol, rely on the generation of a single-cell suspension (which can be visually confirmed during counting). Second, an excessively slow growth rate may considerably extend the duration of the assay and potentially result in an imprecise estimation of radiosensitivity. It is indeed conceivable that an extended culture time as required for slow-growing PDTOs to reach an endpoint-compatible size may offer extra chances for sublethally irradiated PDTO-forming cells to recover from stress and restore detectable (though slow-growing) PDTOs. Finally, dissociating PDTOs shortly after irradiation is expected to impose added stress on PDTO-forming cells, potentially increasing radiosensitivity as a result of altered cell-cell communications in the early phases of recovery. Delaying dissociation of 24-72 h may provide insights into the relative contribution of preserved cell-cell communications to the ability of PDTO-forming cells to recover from macromolecular damage as imposed by RT.
While live imaging platforms offer a convenient method to monitor PDTO formation and growth longitudinally, single endpoint images can also be used for the same objective. In this case, attention should be paid to ensure that adjacent PDTOs do not merge prior to imaging, and growth rate can be assessed as a function of PDTO size normalized to the duration of the assay.
In the era of personalized cancer medicine, assessing the sensitivity of individual tumors to treatment is of the utmost importance as it provides some hints for the design of individualized therapeutic strategies with improved efficacy29. The protocol described herein fits into this endeavor by providing a convenient approach to estimating the cellular sensitivity of individual PDTOs to ionizing radiation (alone or combined with other therapeutic modalities). That said, whether measuring cancer cell radiosensitivity from PDTOs accurately predicts the response of individual patients with cancer to focal RT remains to be demonstrated. Correlating the radiosensitivity of distinct PDTOs to irradiation with the clinical response to RT of their respective donors will provide important insights into this possibility.
The authors have nothing to disclose.
We thank Raymond Briones and Wen H. Shen (Weill Cornell Medical College, New York, NY, USA) for their help with the development of this protocol. This work has been supported by a Transformative Breast Cancer Consortium Grant from the US DoD BCRP (#W81XWH2120034, PI: Formenti).
40 µm mesh filter | Thomas Scientific | 1164H35 | |
B27 | Invitrogen | 17504-044 | |
Cellometer Auto T4 Bright Field Cell Counter | Nexcelom | ||
DMEM F/12 | Corning | 12634-010 | |
Epidermal Growth Factor hEGF | Peprotech | AF-100-15 | |
EVOS FL Digital Inverted Fluorescence Microscope | Thermo Fisher Scientific | 12-563-460 | |
FGF10 | Peprotech | 100-26 | |
FGF7 | Peprotech | 100-19 | |
GlutaMax | Invitrogen | 35050061 | |
Hepes | Invitrogen | 15630-080 | |
IncuCyte software 2021A | Sartorius | version: 2021A | |
Incucyte SX1 | Sartorius | model SX1 | |
Incucyte validated 48 well plate | Corning | 3548 | |
Matrigel | Discovery Labware | 354230 | |
nAc | Sigma Aldrich | A9165-5G | |
Nicotinamide | Sigma-Aldrich | N0636 | |
Noggin | Purchased from the Englander Institute for Precision Medicine, Weill Cornell, NY, USA | ||
Non-treated 6 well plate | Cellstar | 657 185 | |
NR (Heregulin) | Peprotech | 100-03 | |
p38 MAP inhibitor p38i SB202190 | Sigma Aldrich | S7067 | |
PBS | Corning | 21-040-CV | |
PenStrep | Invitrogen | 15140-122 | |
Primocin | Invivogen | ant-pm-1 | |
Rspondin Media | Purchased from the Englander Institute for Precision Medicine, Weill Cornell, NY, USA | ||
Small Animal Radiation Research Platform (SARRP) | Xstrahl Ltd | ||
TGFbeta Receptor Inhibitor A83-01 | Tocris | 2939 | |
Trypan blue Stain (0.4%) | Gibco | 15250-61 | |
TrypLE | Gibco | 112605-028 | |
Y-27632 (RhoKi) | Selleck | S1049 |