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Objective To evaluate the impact of daily fraction doses on late genitourinary (GU) toxicity after salvage radiotherapy (SRT) for prostate cancer. Methods This multi-institutional retrospective study included 212 patients who underwent SRT between 2008 and 2018. All patients received image-guided intensity-modulated SRT at a median dose of 67.2 Gy in 1.8-2.3 Gy/fraction. The cumulative rates of late grade ≥2 GU and gastrointestinal (GI) toxicities were compared using Gray test, stratified by the ≤2.0 Gy/fraction (n  =   137) and ≥2.1 Gy/fraction groups (n  =   75), followed by multivariate analyses. The total dose was represented as an equivalent dose in 2-Gy fractions (EQD2) with α/β = 3 Gy. Results After a median follow-up of 63 months, the cumulative rates of 5-year late grade ≥2 GU and GI toxicities were 14% and 2.5%, respectively. The cumulative rates of 5-year late grade ≥2 GU toxicity in the ≥2.1 Gy/fraction and ≤2.0 Gy/fraction groups were 22% and 10%, respectively (P  =  .020). In the multivariate analysis, ≥2.1 Gy/fraction was still associated with an increased risk of late grade ≥2 GU toxicity (hazard ratio, 2.37; 95% confidence interval, 1.12-4.99; P  =  .023), while the total dose was not significant. Conclusion The present results showed that ≥2.1 Gy/fraction resulted in a higher incidence of late grade ≥2 GU toxicity in SRT. Advances in knowledge The impact of fraction doses on late GU toxicity after SRT remains unknown. The results suggest that higher fraction doses may increase the risk of late GU toxicity in SRT. [ABSTRACT FROM AUTHOR]

Abstract Background Organizing pneumonia (OP), so called bronchiolitis obliterans organizing pneumonia after postoperative irradiation for breast cancer has been often reported. There is little information about OP after other radiation modalities. This cohort study investigated the clinical features and risk factors of OP after stereotactic ablative radiotherapy of the lung (SABR). Methods Patients undergoing SABR between 2004 and 2010 in two institutions were investigated. Blood test and chest computed tomography were performed at intervals of 1 to 3 months after SABR. The criteria for diagnosing OP were: 1) mixture of patchy and ground-glass opacity, 2) general and/or respiratory symptoms lasting for at least 2 weeks, 3) radiographic lesion in the lung volume receiving Results Among 189 patients (164 with stage I lung cancer and 25 with single lung metastasis) analyzed, nine developed OP. The incidence at 2 years was 5.2% (95% confidence interval; 2.6-9.3%). Dyspnea were observed in all patients. Four had fever. These symptoms and pulmonary infiltration rapidly improved after corticosteroid therapy. Eight patients had presented with symptomatic radiation pneumonitis (RP) around the tumor 2 to 7 months before OP. The prior RP history was strongly associated with OP (hazard ratio 61.7; p = 0.0028) in multivariate analysis. Conclusions This is the first report on OP after SABR. The incidence appeared to be relatively high. The symptoms were sometimes severe, but corticosteroid therapy was effective. When patients after SABR present with unusual pneumonia, OP should be considered as a differential diagnosis, especially in patients with prior symptomatic RP.

Abstract Background Radiation proctitis after intensity-modulated radiation therapy (IMRT) differs from that seen after pelvic irradiation in that this adverse event is a result of high-dose radiation to a very small area in the rectum. We evaluated the results of treatment for hemorrhagic proctitis after IMRT for prostate cancer. Methods Between November 2004 and February 2010, 403 patients with prostate cancer were treated with IMRT at 2 institutions. Among these patients, 64 patients who developed late rectal bleeding were evaluated. Forty patients had received IMRT using a linear accelerator and 24 by tomotherapy. Their median age was 72 years. Each patient was assessed clinically and/or endoscopically. Depending on the severity, steroid suppositories or enemas were administered up to twice daily and Argon plasma coagulation (APC) was performed up to 3 times. Response to treatment was evaluated using the Rectal Bleeding Score (RBS), which is the sum of Frequency Score (graded from 1 to 3 by frequency of bleeding) and Amount Score (graded from 1 to 3 by amount of bleeding). Stoppage of bleeding over 3 months was scored as RBS 1. Results The median follow-up period for treatment of rectal bleeding was 35 months (range, 12–69 months). Grade of bleeding was 1 in 31 patients, 2 in 26, and 3 in 7. Nineteen of 45 patients (42%) observed without treatment showed improvement and bleeding stopped in 17 (38%), although mean RBS did not change significantly. Eighteen of 29 patients (62%) treated with steroid suppositories or enemas showed improvement (mean RBS, from 4.1 ± 1.0 to 3.0 ± 1.8, p = 0.003) and bleeding stopped in 9 (31%). One patient treated with steroid enema 0.5-2 times a day for 12 months developed septic shock and died of multiple organ failure. All 12 patients treated with APC showed improvement (mean RBS, 4.7 ± 1.2 to 2.3 ± 1.4, p Conclusions After adequate periods of observation, steroid suppositories/enemas are expected to be effective. However, short duration of administration with appropriate dosage should be appropriate. Even when patients have no response to pharmacotherapy, APC is effective.

Abstract Background In stereotactic body radiotherapy (SBRT) for lung tumors, reducing tumor movement is necessary. In this study, we evaluated changes in tumor movement and percutaneous oxygen saturation (SpO2) levels, and preliminary clinical results of SBRT using the BodyFIX immobilization system. Methods Between 2004 and 2006, 53 consecutive patients were treated for 55 lesions; 42 were stage I non-small cell lung cancer (NSCLC), 10 were metastatic lung cancers, and 3 were local recurrences of NSCLC. Tumor movement was measured with fluoroscopy under breath holding, free breathing on a couch, and free breathing in the BodyFIX system. SpO2 levels were measured with a finger pulseoximeter under each condition. The delivered dose was 44, 48 or 52 Gy, depending on tumor diameter, in 4 fractions over 10 or 11 days. Results By using the BodyFIX system, respiratory tumor movements were significantly reduced compared with the free-breathing condition in both craniocaudal and lateral directions, although the amplitude of reduction in the craniocaudal direction was 3 mm or more in only 27% of the patients. The average SpO2 did not decrease by using the system. At 3 years, the local control rate was 80% for all lesions. Overall survival was 76%, cause-specific survival was 92%, and local progression-free survival was 76% at 3 years in primary NSCLC patients. Grade 2 radiation pneumonitis developed in 7 patients. Conclusion Respiratory tumor movement was modestly suppressed by the BodyFIX system, while the SpO2 level did not decrease. It was considered a simple and effective method for SBRT of lung tumors. Preliminary results were encouraging.

Aim: We aimed to identify the optimal candidates for early salvage radiotherapy (SRT) among patients with biochemical recurrence (BCR) after radical prostatectomy (RP). Methods: This multi‐institutional retrospective study included 371 patients treated using SRT after RP. The median (range) PSA level at BCR was 0.36 (0.10‐2.00) ng/mL. The association between early SRT (ie, starting PSA level < 0.50) and BCR after SRT was tested in each subgroup according to our own risk stratification. Results: The median follow‐up time was 51 months. By multivariate analysis, pT3b, Gleason score ≥ 8, negative surgical margins, PSA doubling time < 6 months, and non‐early SRT were associated with BCR after SRT. Patients were stratified by four risk factors other than non‐early SRT: (1) low risk (0 risk factor), (2) intermediate risk (1 risk factor), and (3) high risk (≥2 risk factors). The BCR‐free survival was higher in the early SRT group than the nonearly SRT group in the high‐risk subgroup (P = 0.020), whereas that was similar between two groups in the low‐risk and intermediate‐risk subgroups (P =.79 and.18, respectively). Multivariate analysis revealed that early SRT was beneficial for the high‐risk subgroup (P =.032), whereas early SRT was not associated with improved outcomes in the low‐risk and intermediate‐risk subgroups (P =.92 and 1.0, respectively). Conclusions: This study suggested that early SRT seemed to contribute to better biochemical control for patients with more adverse features, whereas no benefit was observed in men with no adverse features. [ABSTRACT FROM AUTHOR]

Background: In stereotactic body radiotherapy (SBRT) for lung tumors, reducing tumor movement is necessary. In this study, we evaluated changes in tumor movement and percutaneous oxygen saturation (SpO2) levels, and preliminary clinical results of SBRT using the BodyFIX immobilization system. Methods: Between 2004 and 2006, 53 consecutive patients were treated for 55 lesions; 42 were stage I non-small cell lung cancer (NSCLC), 10 were metastatic lung cancers, and 3 were local recurrences of NSCLC. Tumor movement was measured with fluoroscopy under breath holding, free breathing on a couch, and free breathing in the BodyFIX system. SpO2 levels were measured with a finger pulseoximeter under each condition. The delivered dose was 44, 48 or 52 Gy, depending on tumor diameter, in 4 fractions over 10 or 11 days. Results: By using the BodyFIX system, respiratory tumor movements were significantly reduced compared with the free-breathing condition in both craniocaudal and lateral directions, although the amplitude of reduction in the craniocaudal direction was 3 mm or more in only 27% of the patients. The average SpO2 did not decrease by using the system. At 3 years, the local control rate was 80% for all lesions. Overall survival was 76%, cause-specific survival was 92%, and local progression-free survival was 76% at 3 years in primary NSCLC patients. Grade 2 radiation pneumonitis developed in 7 patients. Conclusion: Respiratory tumor movement was modestly suppressed by the BodyFIX system, while the SpO2 level did not decrease. It was considered a simple and effective method for SBRT of lung tumors. Preliminary results were encouraging. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the relationship between waiting time (WT) and disease progression in patients undergoing stereotactic body radiotherapy (SBRT) for lung adenocarcinoma (AD) or squamous cell carcinoma (SQ). Methods and Materials: 201 patients with Stage I AD or SQ undergoing SBRT between January 2004 and June 2010 were analyzed. The WT was defined as the interval between diagnostic computed tomography before referral and computed tomography for treatment planning or positioning before SBRT. Tumor size was measured on the slice of the longest tumor diameter, and tumor volume was calculated from the longest diameter and the diameter perpendicular to it. Changes in tumor volume and TNM stage progression were evaluated, and volume doubling time (VDT) was estimated. Results: The median WT was 42 days (range, 5-323 days). There was a correlation between WT and rate of increase in volume in both AD and SQ. The median VDTs of AD and SQ were 170 and 93 days, respectively. Thirty-six tumors (23%) did not show volume increase during WTs >25 days. In 41 patients waiting for ≤4 weeks, no patient showed T stage progression, whereas in 25 of 120 (21%) patients waiting for >4 weeks, T stage progressed from T1 to T2 (p = 0.001). In 10 of 110 (9.1%) T1 ADs and 15 of 51 (29%) T1 SQs, T stage progressed (p = 0.002). N stage and M stage progressions were not observed. Conclusion: Generally, a WT of ≤4 weeks seems to be acceptable. The WT seems to be more important in SQ than in AD. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the reliability of the linear-quadratic (LQ) formalism and the magnitude of errors associated with its use in assessing biologic equivalence between single, high radiation doses and hypofractionated radiation doses. Methods and Materials: V79 and EMT6 single cells received single doses of 2–12 Gy or two or three fractions of 4 or 5 Gy, each at 4-h intervals. Single and fractionated doses to actually reduce the cell survival to the same level were determined by a colony assay. The α/β ratio was obtained from the cell survival curves. Using the α/β ratio and the LQ formalism, equivalent single doses for the hypofractionated doses were calculated. They were then compared with the actually determined equivalent single doses for the hypofractionated doses. The V79 spheroids received single doses of 5–26 Gy or two to five fractions of 5–12 Gy at 2 or 4-h interval, and then were assayed for cell survival. Next, equivalent single doses for the hypofractionated doses were determined, as were done for the single cells. Results: The α/β ratio was 5.1 Gy for the V79 single cells and 0.36 Gy for EMT6. In V79, the equivalent single doses for the hypofractionated doses calculated using the LQ formalism were 12–19% lower than the actually measured biologically equivalent single doses. In the EMT6 cells, this trend was also seen, but the differences were not significant. In the V79 spheroids, the calculated doses were 18–30% lower than the measured doses. Conclusion: Conversion of hypofractionated radiation doses to single doses using the LQ formalism could underestimate the effect of hypofractionated radiation by ≤30%. [Copyright &y& Elsevier]

Introduction: Radiation therapy (RT) for choroidal metastasis (CM) aims to preserve vision and achieve local control (LC), thereby maintaining quality of life. The present study reports the clinical outcomes of RT for CM and reviews the literature., Methods: We retrospectively collected data on 11 patients with CM; their primary tumors were breast cancer (n=3), lung cancer (n=3), leukemia (n=2), lymphoma (n=2), and gastric cancer (n=1). Four patients had bilateral CM. The median radiation dose was 39 Gy in 13 fractions (range, 20-50 Gy in 10-25 fractions). We investigated changes in visual acuity, tumor responses, morbidities, LC, and overall survival (OS). A systematic review of literature published between 1990 and 2020 was performed using the PubMed database., Results: One, 1, and 6 patients had improved, stabilized, and worse visual acuity, respectively (data missing for 3 patients). Nevertheless, eight patients considered their visual acuity to have improved or remained the same after RT. Among 15 lesions in 11 patients, complete and partial responses were observed in 2 and 6, respectively (data missing for 7 lesions in 4 patients). Three-year LC and OS rates were 100 and 32%, respectively. Grade ≥ 3 morbidities were not observed. In the literature review, the most common primary cancer was breast cancer followed by lung cancer. Improvements in or the stabilization of visual acuity was observed in 80% of patients (range, 47-100), and the median survival time was 11 months (range, 4.9-23)., Conclusion: RT is an efficient and safe palliative treatment for CM without severe toxicity.

Aim: We aimed to identify the optimal candidates for early salvage radiotherapy (SRT) among patients with biochemical recurrence (BCR) after radical prostatectomy (RP)., Methods: This multi-institutional retrospective study included 371 patients treated using SRT after RP. The median (range) PSA level at BCR was 0.36 (0.10-2.00) ng/mL. The association between early SRT (ie, starting PSA level < 0.50) and BCR after SRT was tested in each subgroup according to our own risk stratification., Results: The median follow-up time was 51 months. By multivariate analysis, pT3b, Gleason score ≥ 8, negative surgical margins, PSA doubling time < 6 months, and non-early SRT were associated with BCR after SRT. Patients were stratified by four risk factors other than non-early SRT: (1) low risk (0 risk factor), (2) intermediate risk (1 risk factor), and (3) high risk (≥2 risk factors). The BCR-free survival was higher in the early SRT group than the nonearly SRT group in the high-risk subgroup (P = 0.020), whereas that was similar between two groups in the low-risk and intermediate-risk subgroups (P = .79 and .18, respectively). Multivariate analysis revealed that early SRT was beneficial for the high-risk subgroup (P = .032), whereas early SRT was not associated with improved outcomes in the low-risk and intermediate-risk subgroups (P = .92 and 1.0, respectively)., Conclusions: This study suggested that early SRT seemed to contribute to better biochemical control for patients with more adverse features, whereas no benefit was observed in men with no adverse features., (© 2020 John Wiley & Sons Australia, Ltd.)

Whole-brain radiotherapy (WBRT) has been an important component of treatment for primary central nervous system lymphoma (PCNSL), but delayed neurotoxicity has been a matter of concern. We have employed partial-brain radiotherapy (PBRT) with wide margins for PCNSL patients with a single lesion or a few lesions. In this study, we evaluated the treatment outcome in PCNSL patients undergoing PBRT. Between 2003 and 2014, 24 patients were treated with PBRT; 16 received high-dose-methotrexate (MTX) -containing chemotherapy before PBRT. Conventional fractionation with a median dose of 54 Gy was used. For reference, 15 patients undergoing MTX-based chemotherapy and WBRT were also analyzed. The 3-year overall survival rate was 60% for all 24 patients undergoing PBRT and 68% for the 16 patients undergoing MTX-based chemotherapy plus PBRT. The 3-year progression-free survival rate was 41% for all 24 patients undergoing PBRT and 36% for the 16 patients undergoing MTX-based chemotherapy. The in-field recurrence rate was 26% and the out-of-field recurrence rate was 15% at 3 years for all 24 patients undergoing PBRT. The rates for in-field recurrence and the out-of-field recurrence were 27% and 21%, respectively, for the 16 patients undergoing MTX-based chemotherapy. CNS-recurrence rates were similar in patients undergoing MTX-based chemotherapy and PBRT to the rates in those undergoing MTX-based chemotherapy and WBRT. Neurocognitive dysfunction developed in 3 of the 16 patients undergoing MTX + PBRT and in 4 of 15 patients undergoing MTX + WBRT (P = 0.68). PBRT seems to be a feasible treatment option for solitary PCNSL. Further investigations are warranted to evaluate the advantages of PBRT over WBRT., (© The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

Outcomes of three protocols of intensity-modulated radiation therapy (IMRT) for localized prostate cancer were evaluated. A total of 259 patients treated with 5-field IMRT between 2005 and 2011 were analyzed. First, 74 patients were treated with a daily fraction of 2.0 Gy to a total of 74 Gy (low risk) or 78 Gy (intermediate or high risk). Then, 101 patients were treated with a 2.1-Gy daily fraction to 73.5 or 77.7 Gy. More recently, 84 patients were treated with a 2.2-Gy fraction to 72.6 or 74.8 Gy. The median patient age was 70 years (range, 54-82) and the follow-up period for living patients was 47 months (range, 18-97). Androgen deprivation therapy was given according to patient risk. The overall and biochemical failure-free survival rates were, respectively, 96 and 82% at 6 years in the 2.0-Gy group, 99 and 96% at 4 years in the 2.1-Gy group, and 99 and 96% at 2 years in the 2.2-Gy group. The biochemical failure-free rate for high-risk patients in all groups was 89% at 4 years. Incidences of Grade ≥ 2 acute genitourinary toxicities were 9.5% in the 2.0-Gy group, 18% in the 2.1-Gy group, and 15% in the 2.2-Gy group (P = 0.29). Cumulative incidences of Grade ≥ 2 late gastrointestinal toxicity were 13% in the 2.0-Gy group at 6 years, 12% in the 2.1-Gy group at 4 years, and 3.7% in the 2.2-Gy group at 2 years (P = 0.23). So far, this stepwise shortening of treatment periods seems to be successful.

A dendritic cell (DC)-based vaccine was combined with intensity-modulated radiotherapy (IMRT) or other conformal radiotherapy (RT), assuming minimal immunosuppression by such RT modalities. In this study, the outcomes in the first 40 patients are presented. The patients had recurrent, metastatic or locally advanced tumors. Nine had previously undergone full-course RT. Peripheral blood mononuclear cells obtained by leukapheresis were cultured with granulocyte-macrophage colony-stimulating factor, interleukin-4, OK-432 and prostaglandin E2 to generate DCs, which were pulsed with autologous tumor lysates or tumor-specific peptides, such as WT1. IMRT using tomotherapy, stereotactic irradiation or 3-dimensional conformal RT (3DCRT) was initially administered. The standard dose was 30 and 60 Gy in patients with and without previous RT, respectively. Every other week thereafter, up to a total of 7 times, DC vaccines were injected directly into the tumor (n=15) or administered intradermally when DCs were pulsed with tumor lysates or peptides. The tumor response was evaluated according to the response evaluation criteria in solid tumors (RECIST). RT and DC vaccines were well tolerated and there were no major complications. Three patients were not able to complete the planned DC therapy due to disease progression. For the 31 patients receiving full-dose RT, the response rate was 61% and for the 9 patients who had previously received RT, the response rate was 55%. In 9 patients, the tumor response outside the RT target volume was evaluable: 22% had a partial response (PR), 33% had stable disease (SD) and 44% had progressive disease (PD). In conclusion, a combination of IMRT (or 3DCRT) and DC vaccine is feasible and requires further investigation.

To ascertain whether meloxicam used in a clinical setting as a non-steroidal anti-inflammatory drug (NSAID) warrants preclinical in vivo evaluation as an anticancer agent, we investigated its antitumor effects alone and in combination with radiation and/or 5-fluorouracil (5-FU) in cultured cells. Seven cell lines were examined for cyclooxygenase-2 (COX-2) protein expression by immunoblot analysis, and the HeLaS3, SCCVII and EMT6 cell lines were selected, expressing relatively high, intermediate, and relatively low COX-2 levels, respectively. Antitumor effects were examined using a colony assay. Among the three cell lines, the effect of meloxicam alone was strongest in SCCVII cells. With 24 h of drug exposure, meloxicam at concentrations of 250 and 1250 µM had a definite antitumor effect, dependent on the drug exposure time. The effect of meloxicam in combination with radiation and/or 5-FU was also investigated in the SCCVII cells. At a meloxicam concentration of 250 µM, the antitumor effect in combination with radiation or 5-FU was increased compared to the effect of radiation or 5-FU alone; however, the combined effect appeared to be additive. At lower concentrations, meloxicam had no radiosensitizing effect, nor did it enhance the effect of 5-FU. A meloxicam concentration of 250 µM is considerably higher than concentrations obtained in humans taking meloxicam as an NSAID. In conclusion, the antitumor effect of meloxicam was not correlated with the level of COX-2 protein expression. The effect of meloxicam in combination with radiation and/or 5-FU appeared to be additive. To evaluate the possibility of using meloxicam as an anticancer agent, in vivo investigations at clinically relevant drug dose levels are required.

The effect of pretreatment with various low doses of total-body irradiation (TBI) on tumor cell transplantability in syngeneic mice was investigated. Two cell lines, EMT6 and SCCVII, and two strains of mice, were used. First, Balb/c mice were sham-irradiated or irradiated at 200 mGy, and 6-48 h later, 1000 EMT6 cells were inoculated in the hind legs. Based on the results, 0-1500 mGy of TBI was given 6 h before inoculation of 100 or 1000 cells in the subsequent experiments. All mice were observed for 50 days after transplantation. Tumors were judged as grown when the volume of palpable nodules exceeded 200 mm(3). Tumor transplantability rate was significantly higher in the groups irradiated at 1500 mGy than in the sham-irradiated groups in both Balb/c and C3H/He mice. There were no differences in transplantability rates between the control group and the groups irradiated at various doses of 50-500 mGy. However, the mean time to tumor appearance was significantly elongated in Balb/c mice receiving TBI at 200 mGy and inoculated with 100 or 1000 EMT6 cells 6 h later. This phenomenon was also observed in Balb/c mice receiving 100 mGy TBI and inoculated with 1000 EMT6 cells. The present study might suggest that low-dose TBI to mice may delay tumor growth under certain conditions.

Radioadaptive survival responses after relatively low doses of radiation were investigated in C57BL/6 mice. The 8-week-old mice received whole-body mid-lethal challenging irradiation (5.9 Gy) at various intervals after conditioning whole-body irradiation with 50-400 mGy. Thereafter, survival of the mice was observed for 30 days. The mice receiving 400 mGy at 6 h before the challenging dose had a lower survival rate than the control group, but it was not observed when the conditioning 400-mGy irradiation was given 24 h beforehand. The conditioning doses of 100 and 200 mGy did not influence the survival of mice after the challenging dose. The mice receiving 50 mGy at 1 day, 3 days or 1 week before the challenging dose had a higher survival rate than the control, although this adaptive response was not observed when 50 mGy was given 6 h, 12 h, 3.5 weeks, or 5 weeks beforehand. When 50 mGy was given 2 weeks before the challenging dose, the adaptive response was observed in an experiment in which the mice were caged in our laboratory at the age of 5 weeks, whereas it was not observed in another experiment in which the mice were caged at 3 weeks. This study confirmed the presence of radioadaptive survival responses at the dose of 50 mGy given relatively shortly before the challenging dose.

The purpose of this study was to investigate radioadaptive response in 4 cell-lines under identical conditions using a colony assay. First, 4 cell-lines (V79, HeLa S3, EMT6 and SCCVII) were exposed to 8 Gy at various intervals after pretreatment with an adapting dose of 50 mGy or without it. Second, V79 cells were exposed to 8 Gy at 4.5 hrs after an adapting dose of 0 to 400 mGy. Third, V79 cells were exposed to 2, 4 or 6 Gy at 6 hrs after an adapting dose of 0 or 50 mGy. In the last experiment, an adapting dose was given either immediately after cell plating or 24 hrs later. Cell survival was assessed by a standard colony assay. Adaptive response was not observed in any of the 4 lines tested. In V79 cells, no adaptive response was seen even by changing the adapting dose, challenging dose, and timing of adapting radiation after cell plating. Although radioadaptive response has been reported for the V79 cell-line, we could not reproduce the result. We also failed to demonstrate the phenomenon in the other 3 tumor cell-lines in culture.