**2. Methods and materials**

We have published in detail the exact protocols that we have used in these studies (21, 22, 23, 24).

#### **2.1 Cell lines**

We study 3 radioresistant glioblastoma cell lines (U251, T98G and U87) two other lines classified as glioblastoma but are more sensitive (GL-13, JW-1T). We compare them in detail with two human colorectal tumor cell lines (DLD-1 and 19S184), both cells expressing mutTP53 but 19S186 has been abrogated in CDKN1A (p21) and this abrogation while not effecting in vitro radiosensitivity causes increased radiosensitivity in xenograft tumors (Waldemann, 24).

#### **2.2 Cell and culture techniques**

The basic media for colon tumor cell lines was McCoy 5A, supplemented with 10% FBS, 1% penicillin and streptomycin, 1% L-glutamine; Human glioma cell lines were cultured in DMEM/F12 with 10% FBS, 1% L-glutamine and 1% Penicillin and streptomycin. All cells were sub-cultured twice a week to maintain exponential growth.

#### **2.3 Cell survival assay**

Cells were plated ~18 hours before irradiation. Surviving colonies were determined 10-14 days after irradiation depending on the cell line. Cells were stained with crystal violet and colonies counted (>50 cells/colony). Additional plates for each experiment were used as microcolony controls. Special care was taken in dispersing cell cultures to obtain single cell suspensions with high plating efficiencies.

#### **2.4 Irradiation**

Cells were irradiated in complete media in a Gammacell 40 (Nordion Ottawa ONT Canada) at approximately 0.7 Gy/min. Cells were plated 15 to 18 hours before irradiation with careful measurement of plating efficiency and multiplicity. After exposure, plates were incubated for 8-14 days depending on specific growth and colonies stained with crystal violet. Colonies with more than 50 cells were counted. For each cell line we performed

generally not modified by either by dose-rate or ionization density. In contradistinction, the alpha\* and omega\* responses are highly susceptible to dose-rate and ionization density.

**1.6 Coefficients that represent the modulation of in vivo radiosensitivity by genotype** 

We demonstrated variation in the response of tumor xenografts to radiotherapy protocols based on genotype and dose-schedule. In these studies, Williams et al 21, we showed genotype of tumor cells influenced both in vitro radiosensitivity of tumor cells and also, by a different mechanism, influenced xenograft response in vivo. We attributed this effect, that was substantial in some cells, as an interaction between tumor genotype and the in vivo tumor microenvironment. Importantly one glioblastoma line that was in the VR cellular radiosensitivity group, expressed surprising sensitivity when irradiated as xenograft tumors

We have published in detail the exact protocols that we have used in these studies (21, 22,

We study 3 radioresistant glioblastoma cell lines (U251, T98G and U87) two other lines classified as glioblastoma but are more sensitive (GL-13, JW-1T). We compare them in detail with two human colorectal tumor cell lines (DLD-1 and 19S184), both cells expressing mutTP53 but 19S186 has been abrogated in CDKN1A (p21) and this abrogation while not effecting in vitro radiosensitivity causes increased radiosensitivity in xenograft tumors

The basic media for colon tumor cell lines was McCoy 5A, supplemented with 10% FBS, 1% penicillin and streptomycin, 1% L-glutamine; Human glioma cell lines were cultured in DMEM/F12 with 10% FBS, 1% L-glutamine and 1% Penicillin and streptomycin. All cells

Cells were plated ~18 hours before irradiation. Surviving colonies were determined 10-14 days after irradiation depending on the cell line. Cells were stained with crystal violet and colonies counted (>50 cells/colony). Additional plates for each experiment were used as microcolony controls. Special care was taken in dispersing cell cultures to obtain single cell

Cells were irradiated in complete media in a Gammacell 40 (Nordion Ottawa ONT Canada) at approximately 0.7 Gy/min. Cells were plated 15 to 18 hours before irradiation with careful measurement of plating efficiency and multiplicity. After exposure, plates were incubated for 8-14 days depending on specific growth and colonies stained with crystal violet. Colonies with more than 50 cells were counted. For each cell line we performed

were sub-cultured twice a week to maintain exponential growth.

**and dose** 

in vivo.

23, 24).

**2.1 Cell lines** 

(Waldemann, 24).

**2.3 Cell survival assay** 

**2.4 Irradiation** 

**2. Methods and materials** 

**2.2 Cell and culture techniques** 

suspensions with high plating efficiencies.

controls to account for possible proliferation during the period between plating and irradiation. This control consisted of plating 105 cells in separate plates when replicates of cells were plated for colony formation. When irradiation was performed on the plates for colony formation, the microcolony plates were stained and the number of cells per colony measured. The average number of cells per colony was below 1.20 cells per microcolony for all cell lines and did not vary significantly between cell lines.

Low dose rate irradiation was carried out in a specially constructed Cs-137 irradiator with temperature control and the ability to irradiate cells with constant or exponentiallydecreasing dose-rates.

#### **2.5 Regrowth delay in xenograft tumors**

Tumors were established by subcutaneous injection of 5 million cells suspended in PBS into the upper thigh of nude mice. Each cohort included 6 to 13 tumors. Tumor growth rate was determined by measuring three orthogonal diameters of each tumor twice a week and the tumor volume estimated as π/6 [D1 x D2 x D3], when individual tumor volumes reached ~0.1-0.3 cm3, radiation treatment was initiated. Modal specific growth delay (mSGD) was measured for all cohorts in which a majority of tumors reached a volume four times the initial volume. Response was normalized to growth of unirradiated cells. We chose not to use the mean of specific regrowth delay patterns since a significant proportion of our cohorts included one or more tumors that did not regrow. Thus the mean became limited as a regrowth parameter. For cohorts for which some tumors did not regrow we estimated mSGD based on the regrowth pattern for the minority of tumors that did regrow. When we tested the sensitivity of modal to mean growth delay in selected cohorts in which all tumors regrew, the modal value always fell within one standard deviation of the mean. These methods share some characteristics of the methods described by Schwatchofer [25]. To provide an overview of the dichotomous response when some tumors regrow but some do not, we indicated such cohorts with an arrow showing this value, in terms of overall tumor response, was the common minimum response.

#### **3. Analyses**

#### **3.1 Clonogenic inactivation of radioresistant glioblastoma cell lines**

In our previous studies (21, 22) we identified three glioblastoma cell lines (U251, T98G, U-87) that were the most resistant of 39 cell lines examined as defined by comparison of clonogenic inactivation between circa 2 Gy and 10 Gy. These three radioresistant cell lines expressed two forms of TP53, with U251 and T98 expressing mutTP53 and U87 expressing wtTP53. For designation purposes we will refer to these three cell lines as expressing a VR radiosensitivity phenotype and expressing either a glio+mutTP53 genotype (U251 and T98G) or a glio+wtTP53 genotype. In figure 1 we compare clonogenic inactivation curves for these three VR (very radioresistant) glioblastoma cell lines compared to two colorectal cancer cell lines that fall into the R (radioresistant) radiosensitivity group wtTP53 (HCT116) and its subline abrogated in p21 (19S186) .

The data in figure 1 show relative radiosensitivity between the five cell lines but it is important in our interpretation of these data to show them in the context of overall radiosensitivity of human tumor cell lines. In figure 1 there are clear differences between the three glioblastoma cell lines and the two more sensitive colorectal tumors. These differences vary with the dose-segment over which the data are presented.

Radiobiology of Radioresistant Glioblastoma 11

In the bottom left panel the responses of all five cell lines are shown for their detailed responses over doses between 0.0 and 0.20 Gy. In this panel, the dashed lines are the slopes for each cell line defined by connecting 0.0 dose points to the points at 0.10 Gy, α (SF.1),

All cell lines change in their rates of inactivation at 0.10 Gy that represents the induction of the R response. Note that the rates of inactivation over the R responses (0.1 to 0.2 Gy), varies between cell lines with U87 showing a marked increase compared to the other four lines. . In the lower right panel, the rates of inactivation over the alpha\* responses are shown. The rates for the slopes of the alpha\* response are calculated by the slopes between 0.20 and 2.0 Gy indicated as α (.2-2.). The slopes of the For this dose-segment, all three VR cell lines are

In a larger cohort of cells we have previously shown that the alpha responses of the three VR lines are distinctly more resistant (22). In our studies of multiple components (20) we showed that the alpha response is comprised of the "average" slope for the H, R and alpha\* responses. This work also showed there is correlation between the alpha\* response measured over the dose-segment from 0.2 Gy to 2.0 Gy an the general alpha response shown

**3.2 Coefficients that define the alpha and omega responses segregate human tumor** 

In figure 2 we show a scatter diagram based on our data from Williams et al (21) expressed as values of the coefficients derived for the alpha and omega responses measured as shown in figure 1 and measured as the slope of the general alpha response and the omega response. This figure also specifically identifies the five cell lines that are the subject of our present analysis: U251, T98G, U87, DLD-1 and shows they are distinctly different in their radiosensitivity compared to the lines JW-1T and GL-13 purported also to be glioblastoma

There are important implications of these data. First the values of the alpha response and omega response segregate all cell lines into four statistically distinct radiosensitivity groups: VS, S, R and VR. The alpha response is the predominant determinate of radiosensitivity group. Note that the five cell lines that we study in this chapter are distributed in two clusters: U-251 and T98G are clustered in cell lines that express extreme resistance based on their alpha and omega responses. Three cell lines cluster in patterns with the lowest values for both alpha and omega responses, but three cell lines, DLD-1, 19S186 and U-87 while showing resistance to lower doses (alpha response) have significantly larger values of the omega response are also determined as that are also resistant (alpha response) but show elevated values of their omega responses. Hence the three glioblastoma cells in the VR group share the smallest values for their alpha response but vary significantly in their omega responses. Two cell lines classified in the literature as glioblastoma GL-13 and JW-1T fall into distinctly different radiosensitivities

The omega response for U87 cells is distinctly higher, reflecting, we hypothesize, the role of wtTP53 in "glio" cells. We hypothesize that over the alpha response, "glio" confers radioresistance beyond that characterized by expression of mutTP53. At higher doses, wtTP53 modulates radiosensitivity as shown for U87 cells. The data in this figure show

extended to illustrate the strong variation in slope for the H responses.

more resistant than the two R lines.

**cells into four radiosensitivity groups** 

segregating with the S radiosensitivity groups

three distinct clusters of glioblastoma cell lines.

in the upper panel.

cells.

Fig. 1. Radiosensitivity curves (2 to 10 Gy) for five cell lines: three radioresistant human glioblastoma cell lines, U251, T98G, U87, and two human colorectal tumor cell lines, HCT116 and DLD-1. These data show standard survival measurements in the upper panel expressed as two general responses, the α response from 0.0 to circa 3.0 Gy and the ω response for doses greater than circa 3 Gy. The lower two panels show three components that together constitute the alpha response: the H response, the R response and the α\* response.

In the top panel the overall responses are shown between 0 and 10 Gy for the five cell lines and these responses can be analyzed by measuring the slope of inactivation between 0.0 Gy and 2.0 Gy and defined as the alpha response. The values for the alpha response calculated in this manner are significantly higher for VR cells than R cells but these differences are difficult to visualize at the scale used in this panel so the dose-response patterns are expanded in the lower panels.

Similarly an omega response can be calculated for all five cell lines using linear regression of all data points above 4 Gy and the slopes of the five lines do not segregate between the two radiosensitivity groups, with two VR lines U251 and T98G showing a more resistant response than the third line U87. This dichotomy in response corresponds to the differences in these three lines in their expression of TP53. U251 and T98G express mutTP53 while U87 expresses wtTP53. These differences are shown more clearly subsequently.

**Clonal Inactivation of Tumor Cells**

**0 2.5 5 7.5 10 0 to 10 Gy** 

**-0.8**

<sup>α</sup>**\* response -0.5**

the alpha response: the H response, the R response and the α\* response.

α **(SF.1)**

α**\* response** ω **response**

**0 0.1 0.2 0.3 0.4 0.5**

**H T** α**\* response**

**0 - 0.5 Gy**

**-0.4 -0.3 -0.2 -0.1 0**

expanded in the lower panels.

subsequently.

**Log SF**

**Log SF** 

**-2.5 -2.0 -1.5 -1.0 -0.5 0.0**

**(Gy)**

**02U251 T98G U87 DLD-1 19S186**

**Dose** 

In the top panel the overall responses are shown between 0 and 10 Gy for the five cell lines and these responses can be analyzed by measuring the slope of inactivation between 0.0 Gy and 2.0 Gy and defined as the alpha response. The values for the alpha response calculated in this manner are significantly higher for VR cells than R cells but these differences are difficult to visualize at the scale used in this panel so the dose-response patterns are

Similarly an omega response can be calculated for all five cell lines using linear regression of all data points above 4 Gy and the slopes of the five lines do not segregate between the two radiosensitivity groups, with two VR lines U251 and T98G showing a more resistant response than the third line U87. This dichotomy in response corresponds to the differences in these three lines in their expression of TP53. U251 and T98G express mutTP53 while U87 expresses wtTP53. These differences are shown more clearly

Fig. 1. Radiosensitivity curves (2 to 10 Gy) for five cell lines: three radioresistant human glioblastoma cell lines, U251, T98G, U87, and two human colorectal tumor cell lines, HCT116 and DLD-1. These data show standard survival measurements in the upper panel expressed as two general responses, the α response from 0.0 to circa 3.0 Gy and the ω response for doses greater than circa 3 Gy. The lower two panels show three components that together constitute

**-0.6**

**-0.4**

**-0.2**

**0**

**0 0.5 1 1.5 2 2.5**

α **(.2-2)**

**0 - 2.5 Gy**

In the bottom left panel the responses of all five cell lines are shown for their detailed responses over doses between 0.0 and 0.20 Gy. In this panel, the dashed lines are the slopes for each cell line defined by connecting 0.0 dose points to the points at 0.10 Gy, α (SF.1), extended to illustrate the strong variation in slope for the H responses.

All cell lines change in their rates of inactivation at 0.10 Gy that represents the induction of the R response. Note that the rates of inactivation over the R responses (0.1 to 0.2 Gy), varies between cell lines with U87 showing a marked increase compared to the other four lines. .

In the lower right panel, the rates of inactivation over the alpha\* responses are shown. The rates for the slopes of the alpha\* response are calculated by the slopes between 0.20 and 2.0 Gy indicated as α (.2-2.). The slopes of the For this dose-segment, all three VR cell lines are more resistant than the two R lines.

In a larger cohort of cells we have previously shown that the alpha responses of the three VR lines are distinctly more resistant (22). In our studies of multiple components (20) we showed that the alpha response is comprised of the "average" slope for the H, R and alpha\* responses. This work also showed there is correlation between the alpha\* response measured over the dose-segment from 0.2 Gy to 2.0 Gy an the general alpha response shown in the upper panel.

#### **3.2 Coefficients that define the alpha and omega responses segregate human tumor cells into four radiosensitivity groups**

In figure 2 we show a scatter diagram based on our data from Williams et al (21) expressed as values of the coefficients derived for the alpha and omega responses measured as shown in figure 1 and measured as the slope of the general alpha response and the omega response. This figure also specifically identifies the five cell lines that are the subject of our present analysis: U251, T98G, U87, DLD-1 and shows they are distinctly different in their radiosensitivity compared to the lines JW-1T and GL-13 purported also to be glioblastoma cells.

There are important implications of these data. First the values of the alpha response and omega response segregate all cell lines into four statistically distinct radiosensitivity groups: VS, S, R and VR. The alpha response is the predominant determinate of radiosensitivity group. Note that the five cell lines that we study in this chapter are distributed in two clusters: U-251 and T98G are clustered in cell lines that express extreme resistance based on their alpha and omega responses. Three cell lines cluster in patterns with the lowest values for both alpha and omega responses, but three cell lines, DLD-1, 19S186 and U-87 while showing resistance to lower doses (alpha response) have significantly larger values of the omega response are also determined as that are also resistant (alpha response) but show elevated values of their omega responses. Hence the three glioblastoma cells in the VR group share the smallest values for their alpha response but vary significantly in their omega responses. Two cell lines classified in the literature as glioblastoma GL-13 and JW-1T fall into distinctly different radiosensitivities segregating with the S radiosensitivity groups

The omega response for U87 cells is distinctly higher, reflecting, we hypothesize, the role of wtTP53 in "glio" cells. We hypothesize that over the alpha response, "glio" confers radioresistance beyond that characterized by expression of mutTP53. At higher doses, wtTP53 modulates radiosensitivity as shown for U87 cells. The data in this figure show three distinct clusters of glioblastoma cell lines.

Radiobiology of Radioresistant Glioblastoma 13

**(Gy)**

α**(HDR)**

α(**LDR)**

Fig. 3. Comparison of clonogenic inactivation induced by acute high dose-rate HDR (50 Gy/hr) and protracted irradiation LDR (0.25 Gy/hr). The dashed lines represent the

extrapolation of the rate of inactivation at lower doses based on the slopes of inactivation by

**-4**

**-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5**

**-0.3 -0.25 -0.2 -0.15 -0.1 -0.05 0**

**Log(SF) / Gy**

**Log SF**

**HDR (50.0Gy/hr) LDR (0.25Gy)**

**-3**

**-2**

**Log SF**

**-4**

LDR and HDR.

**-3 -2.5 -2 -1.5 -1 -0.5 0 0.5**

**-0.35 -0.3 -0.25 -0.2 -0.15 -0.1 -0.05 0**

**Log(SF) / Gy**

**Log SF**

**-3**

**-2**

**Log SF**

**-1**

**0**

**0 5 10 15 20 25**

**0 5 10 15 20 25**

**U251**

**HDR LDR alphaD (HDR)**

**Dose(Gy)**

**0 12 24 36 48 60 72 84 96**

**Time (hour)**

**omegaD (HDR) -3**

**Log SF/Gy alpha(HDR)**

**omega(HDR) -0.35**

**U251**

**-1**

**0**

**0 5 10 15 20 25**

**0 5 10 15 20 25**

**HDR LDR alphaD (HDR) omegaD (HDR) omegaD (LDR)**

**U87**

**Dose(Gy)**

**0 12 24 36 48 60 72 84 96**

**Time (hour)**

**Log SF/Gy alpha(HDR) omega(HDR)**

**DLD-1**

**(Gy)**

α**(HDR)** α**(LDR)**
