Response of irradiated and bystander cells to charged particles in two-dimensional and three-dimensional colon models

The radiation-induced bystander effect, wherein unirradiated cells near to or sharing medium with irradiated cells express biological responses, most often has been studied in two-dimensional monolayer cultures, although some studies with three-dimensional models and in vivo have also shown bystander signaling. We have shown previously that DNA damage, measured as foci of the DNA repair-related protein 53BP1, occurs in unirradiated bystander cells in a three-dimensional skin epithelium model irradiated with protons or iron ions (Lumpkins et al., submitted). In the current work, we extend the studies to a second epithelial model, colon, with studies in both two-dimensional monolayer and a three-dimensional tissue model using Caco2 human colon epithelial cancer cells and AG01522 human fibroblasts. For the monolayer studies, Caco2 cells in exponential growth were irradiated then co-cultured, sharing medium in an insert system, with unirradiated cells. Cells were irradiated with 250 kVp X-rays at Massachusetts General Hospital or with 1 GeV/amu protons, silicon ions or iron ions at the National Space Radiation Laboratory at Brookhaven National Laboratory. At varying times after irradiation, cell damage was assayed as micronuclei (MN) induction or formation of 53BP1 foci in both irradiated and bystander cells. For the three-dimensional studies, AG01522 fibroblasts were embedded in a collagen gel, then Caco2 cells were grown on the top of the gel. Each three-dimensional construct was cut in half prior to irradiation, with one half irradiated then immediately placed in contact with the other, bystander, half for co-culture. At selected times after irradiation, irradiated and bystander construct halves were fixed and sectioned, and 53BP1 foci were counted. In monolayer culture, irradiated Caco2 cells showed a dose-dependent increased fraction of cells with MN after exposure to X-rays, protons, iron ions or silicon ions. Bystander Caco2 cells sharing medium with the irradiated cells also showed an increased fraction of cells with MN, reaching similar levels of ∼16% cells with MN, about a threefold increase over controls, after 1 Gy of all types of radiation. The fraction of cells with 53BP1 foci depended on radiation type and time of assay after irradiation, with the induction of foci generally greatest 5 h after irradiation and increasing with radiation dose. In bystander Caco2 cells, the appearance of foci generally was delayed, with the maximal fraction of cells showing foci at 12 h. In three-dimensional culture, after X-ray or proton exposure, cells showed similar trends to those seen in two-dimensional growth, i.e. with both the Caco2 and the AG01522 cells, the fraction of irradiated cells having 53BP1 foci reached a maximum at 5 h, but with bystander cells, the maximum occurred at 12 h after irradiation. This delay in the appearance of foci in bystander cells compared with irradiated cells is similar to our findings in the three-dimensional skin model composed of keratinocytes and fibroblasts. In summary, our data now show in two different epithelial tissue models in both two-dimensional and three-dimensional models, radiation-stimulated intercellular signaling results in substantial levels of DNA damage in unirradiated cells. Because Caco2 cells are a carcinoma cell line, the studies are now being extended to a three-dimensional colon model using normal human colonic epithelial cells

Prospective Study of Isolated Recurrent Tumor Re-irradiation With Carbon-Ion Beams

Purpose: To perform a prospective study to evaluate the efficacy and safety of isolated recurrent tumor re-irradiation with carbon-ion radiotherapy (RT).Methods and Materials: The inclusion criteria were clinically proven recurrent tumors, measurable by computed tomography or magnetic resonance imaging, patients ≥ 16 years old, performance status scores between 0 and 2, isolated tumor at a previously irradiated site, and a life expectancy > 6 months. The exclusion criteria were tumor invasion into the gastrointestinal tract or a major blood vessel, uncontrolled infection, early recurrence (<3 months), and severe concomitant diseases. The primary end-point was the local control rate, the secondary end-points including the overall survival rate, and adverse events.Results: Between December 2013 and March 2016, 22 patients were enrolled in this prospective study. All patients were re-irradiated with carbon-ion RT with radical intent. Five patients had rectal cancer, 4 had sarcoma, 4 had lung cancer, 3 had hepatic cell carcinoma, and 6 had other tumors. The median follow-up time was 26 months. Eight patients developed local recurrence, and the 1- and 2-year local control rates were 71 and 60%, respectively. Eight patients died of their cancers and 2 died of other diseases. The 1- and 2-year overall survival rates were 76 and 67%, respectively. There were no grade 2 or higher acute adverse events and 4 patients (18%) developed grade 3 late adverse events. The group with the longer interval (>16 months) between the first RT and re-irradiation had significantly better outcomes than the shorter interval group (≤ 16 months).Conclusions: Re-irradiation, using carbon-ion RT with radical intent, had favorable local control and overall survival rates without severe toxicities for selected patients. Re-irradiation has the potential to improve clinical outcomes for isolated, local, recurrent tumors; further investigations are required to confirm the therapeutic efficacy

Characterization of Torin2, an ATP-Competitive Inhibitor of mTOR, ATM, and ATR

mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival, and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here, we report the characterization of Torin2, a second-generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC[subscript 50] of 250 pmol/L with approximately 800-fold selectivity for cellular mTOR versus phosphoinositide 3-kinase (PI3K). Torin2 also exhibited potent biochemical and cellular activity against phosphatidylinositol-3 kinase–like kinase (PIKK) family kinases including ATM (EC[subscript 50], 28 nmol/L), ATR (EC[subscript 50], 35 nmol/L), and DNA-PK (EC[subscript 50], 118 nmol/L; PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single-agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncologic settings where mTOR signaling has a pathogenic role

Tumour resistance in induced pluripotent stem cells derived from naked mole-rats

The naked mole-rat (NMR, Heterocephalus glaber), which is the longest-lived rodent species, exhibits extraordinary resistance to cancer. Here we report that NMR somatic cells exhibit a unique tumour-suppressor response to reprogramming induction. In this study, we generate NMR-induced pluripotent stem cells (NMR-iPSCs) and find that NMR-iPSCs do not exhibit teratoma-forming tumorigenicity due to the species-specific activation of tumour-suppressor alternative reading frame (ARF) and a disruption mutation of the oncogene ES cell-expressed Ras (ERAS). The forced expression of Arf in mouse iPSCs markedly reduces tumorigenicity. Furthermore, we identify an NMR-specific tumour-suppression phenotype—ARF suppression-induced senescence (ASIS)—that may protect iPSCs and somatic cells from ARF suppression and, as a consequence, tumorigenicity. Thus, NMR-specific ARF regulation and the disruption of ERAS regulate tumour resistance in NMR-iPSCs. Our findings obtained from studies of NMR-iPSCs provide new insight into the mechanisms of tumorigenicity in iPSCs and cancer resistance in the NMR

Probabilistic dose distribution from interfractional motion in carbon ion radiation therapy for prostate cancer shows rectum sparing with moderate target coverage degradation.

PURPOSE:This observational study investigates the influence of interfractional motion on clinical target volume (CTV) coverage, planning target volume (PTV) margins, and rectum tissue sparing in carbon ion radiation therapy (CIRT). It reports dose coverage to target structures and organs at risk in the presence of interfractional motion, investigates rectal tissue sparing, and provides recommendations for lowering the rate of toxicity. We also propose probabilistic DVH based on cone-beam computed tomography (CBCT) table shifts from photon therapy for consideration in bone-matching CIRT treatment planning to represent probable dose to our CIRT patient population. METHODS:At Gunma University Hospital intensity-modulated x-ray therapy (IMXT, aka IMRT) prostate cancer patients are positioned on a table which is shifted twice based on CBCT to align bones and then align prostate tissue to isocenter. These shifts thereby contain interfractional motion. A total of 1306 such table shifts from 85 patients were collected. Normal probability distributions were fit to the difference between bone-matching and prostate-matching CBCT-to-planning CT table shifts (i.e. interfractional motion). Between 2011 and 2016 CIRT prostate patients were treated with three beams to PTV1 (lateral-opposing and anterior) one per day for 9 fractions and two beams for a boost PTV2 (lateral-opposing) one per day for 7 fractions for a prescribed total of 57.6 Gy(RBE) as follows: PTV1 extends the prostate contour by 10/10, 5/10, 6/6 mm in the right/left, posterior/anterior, and superior/inferior directions, respectively, and the proximal seminal vesicles contour by 5 mm superiorly and inferiorly, 3 mm right and left. PTV2 reduces PTV1 posteriorly along a straight line to exclude the rectum and reduces the superior and inferior margins by 6 mm. Probable interfractional motion for 40 patients was simulated using each patient's own beam data as follows: The previously fit normal probability distributions were randomly sampled 2000 times per patient, and the five beams were shifted and summed with the same relative weighting as in the 16-fraction regimen. The resulting dose distribution was then scaled back down by 16/2000 to match the prescribed number of fractions. We then analyzed the resulting doses to contoured structures. RESULTS:Probable dose to rectum is substantially less than planned: For example, mean+-standard deviation D2% for planned and probable DVH is 51+-1.9 and 45+-2.4, respectively. Cumulative DVH show mean CTV fraction receiving a given probable dose is less than the mean fraction receiving the corresponding planned dose for doses larger than 52 Gy(RBE), up to 19% less at 57.4 Gy(RBE). Our PTV1 margins generally cover 95% of interfractional motion but seminal vesicles and inferior prostate receive less dose than planned due to insufficient PTV2 margins. CONCLUSION:Assuming rigidly shifting interfractional motion around the prostate region and neglecting minor changes in soft tissue stopping power, interfractional motion resulted in target underdosing but better tissue sparing in all cases. Given our low rates of relapse and recurrence, it appears less curative dose is needed than previously thought or else current planning target margins may be excessive: Planning target volumes should be reconsidered with the adoption of dose verification methods. Our probable dose distributions quantify expected dose for future dose verification studies

Data from: Probabilistic interfractional motion carbon ion radiation therapy dose distribution for prostate cancer shows rectum sparing with moderate target coverage degradation

Purpose: This observational study investigates the influence of interfractional motion on clinical target volume (CTV) coverage, planning target volume (PTV) margins, and rectum tissue sparing in carbon ion radiation therapy (CIRT). It reports dose coverage to target structures and organs at risk in the presence of interfractional motion, investigates rectal tissue sparing, and provides recommendations for further lowering the rate of toxicity. We also propose probabilistic DVH for consideration in treatment planning to represent probable dose to the clinic’s patient population. Methods: At Gunma University Hospital intensity-modulated x-ray therapy (IMXT, aka IMRT) prostate cancer patients are positioned on a table which is shifted twice based on cone-beam computed tomography (CBCT) to align bones and then align prostate tissue to isocenter. These shifts thereby contain interfractional motion. 1306 such tableshifts from 85 patients were collected. Normal probability distributions were fit to the difference between bone-matching and prostate-matching CBCT-to-planning CT tableshifts (i.e. interfractional motion). Between 2011 and 2016 CIRT prostate patients were treated with PTV1 and PTV2 margins as follows: PTV1 extends the prostate contour by 10/10, 5/10, 6/6 mm in the right/left, posterior/anterior, and superior/inferior directions, respectively, and the proximal seminal vesicles contour by 5 mm superiorly and inferiorly, 3 mm right and left. PTV2 reduces PTV1 posteriorly along a straight line to exclude the rectum and reduces the superior and inferior margins by 6 mm. From those treated with these margins, 40 patients’ beam data were selected to create probable interfractional motion: The previously fit normal probability distributions were randomly sampled 2000 times per patient and beams shifted to simulate this motion. These shifted dose distributions were scaled down proportionately in magnitude and summed to obtain probable blurred dose distributions. Results: Probable dose to rectum is substantially less than planned for doses higher than 10 Gy(RBE). Absolute DVH show that mean clinical target volumes are about 138-670 cm3 smaller for a given probable dose than planned doses higher than 57 Gy(RBE) after accounting for standard error. Cumulative DVH show mean CTV fraction receiving a given probable dose is less than the mean fraction receiving the corresponding planned dose for doses larger than 52 Gy(RBE), up to 19% less at 57.4 Gy(RBE). Our PTV1 margins generally cover 95% of interfractional motion but seminal vesicles and inferior prostate receive less dose than planned due to insufficient PTV2 margins. Conclusion: Assuming rigidly shifting interfractional motion around the prostate region and neglecting minor changes in soft tissue stopping power, interfractional motion resulted in underdosing or tissue sparing in all cases. Given our low rates of relapse and recurrence, it appears less curative dose is needed than previously thought or else the target may be smaller than previously thought. In-room CT may be useful to lower dose, shrink target margins, conduct PET auto-activation dose verification studies and account for interfractional motion

Differential bystander signaling between radioresistant chondrosarcoma cells and fibroblasts after x-ray, proton, iron ion and carbon ion exposures

Purpose\nChondrosarcoma is well known as a radioresistant tumor, but the mechanisms underlying that resistance are still unclear. The bystander effect is well documented in the field of radiation biology. We investigated the bystander response induced by X-rays, protons, carbon ions, and iron ions in chondrosarcoma cells using a transwell insert co-culture system that precludes physical contact between targeted and bystander cells.Methods and Materials\nHuman chondrosarcoma cells were irradiated with 0.1-, 0.5-, 1-, and 2-Gy X-rays, protons, carbon ions or iron ions using a transwell insert co-culture system. Formation of micronuclei and p53 binding protein 1 staining in bystander and irradiated cells were analyzed and bystander signaling between mixed cultures of chondrosarcoma cells, and normal human skin fibroblasts was investigated.Results\nIn this study, we show that the fraction of cells with DNA damages in irradiated chondrosarcoma cells showed dose-dependent increases with all beams. However, the fraction of cells with DNA damages in all bystander chondrosarcoma cells did not show any change from the levels in control cells. In the bystander signaling between mixed cultures of chondrosarcoma cells and fibroblasts, the amount of micronucleus formation in all bystander chondrosarcoma cells co-cultured with irradiated fibroblasts were the same as the levels for control cells. However, all bystander fibroblasts co-cultured with irradiated chondrosarcoma cells showed significant increases in the fraction of micronucleated cells compared to the rate of control cells.Conclusions\nWe conclude that chondrosarcoma cells in the transwell insert co-culture system could release bystander stimulations but could not develop bystander responses

A Promising Treatment Strategy for Lung Cancer: A Combination of Radiotherapy and Immunotherapy

Lung cancer is a leading cause of cancer-related deaths worldwide despite advances in treatment. In the past few decades, radiotherapy has achieved outstanding technical advances and is being widely used as a definitive, prophylactic, or palliative treatment of patients with lung cancer. The anti-tumor effects of radiotherapy are considered to result in DNA damage in cancer cells. Moreover, recent evidence has demonstrated another advantage of radiotherapy: the induction of anti-tumor immune responses, which play an essential role in cancer control. In contrast, radiotherapy induces an immunosuppressive response. These conflicting reactions after radiotherapy suggest that maximizing immune response to radiotherapy by combining immunotherapy has potential to achieve more effective anti-tumor response than using each alone. Immune checkpoint molecules, such as cytotoxic T-lymphocyte-associated protein 4, programmed cell death-1/programmed death-ligand 1, and their inhibitors, have attracted significant attention for overcoming the immunosuppressive conditions in patients with cancer. Therefore, the combination of immune checkpoint inhibitors and radiotherapy is promising. Emerging preclinical and clinical studies have demonstrated the rationale for these combination strategies. In this review, we outlined evidence suggesting that combination of radiotherapy, including particle therapy using protons and carbon ions, with immunotherapy in lung cancer treatment could be a promising treatment strategy