Low-dose XRT Research Articles

Radiation Therapy Modulates Tumor Physical Characteristics to Reduce Intratumoral Pressure and Enhance Intratumoral Drug Delivery and Retention

PurposeHigh intratumoral pressure, caused by tumor cell-to-cell interactions, interstitial fluid pressure, and surrounding stromal composition, plays a substantial role in resistance to intratumoral drug delivery and distribution. Radiation therapy (XRT) is commonly administered in conjunction with different intratumoral drugs, but assessing how radiation can reduce pressure locally and help intratumoral drug administration and retention is important. Methods and Materials344SQ-parental or 344SQ-anti-programmed cell death protein 1-resistant lung adenocarcinoma cells were established in 129Sv/Ev mice, and irradiated with either 1 Gy × 2, 5 Gy × 3, 8 Gy × 3, 12 Gy × 3, or 20 Gy × 1. Intratumoral pressure was measured every 3 to 4 days after XRT. Contrast dye was injected into the tumors 3- and 6-days after XRT, and imaged to measure drug retention. ResultsIn the 344SQ-parental model, low-dose radiation (1 Gy × 2) created an early window of reduced intratumoral pressure 1 to 3 days after XRT compared with untreated control. High-dose stereotactic radiation (12 Gy × 3) reduced intratumoral pressure 3 to 12 days after XRT, and 20 Gy × 1 showed a delayed pressure reduction on day 12. Intermediate doses of radiation did not significantly affect intratumoral pressure. In the more aggressive 344SQ-anti-programmed cell death protein 1-resistant model, low-dose radiation reduced pressure 1 to 5 days after XRT, and 12 Gy × 3 reduced pressure 1 to 3 days after XRT. Moreover, both 1 Gy × 2 and 12 Gy × 3 significantly improved drug retention 3 days after XRT; however, there was no significance detected 6 days after XRT. Lastly, a histopathologic evaluation showed that 1 Gy × 2 reduced collagen deposition within the tumor, and 12 Gy × 3 led to more necrotic core and higher extracellular matrix formation in the tumor periphery. ConclusionsOptimized low-dose XRT, as well as higher stereotactic XRT regimen led to a reduction in intratumoral pressure and increased drug retention. The findings from this work can be readily translated into the clinic to enhance intratumoral injections of various anticancer agents.

NBTXR3 Nanoparticle With Immunoradiation Might Reshape Metastatic Tumor-Infiltrating T Cell Repertoire in Murine Lung Cancer Model

Radiation therapy (XRT) induces lymphocyte infiltration of primary tumors that is increased when using NBTXR3 radioenhancer nanoparticle, leading to improved response to combination immunotherapy (IT). However, treatment effects on the metastatic tumor microenvironment are poorly understood. We studied the effects of NBTXR3 injection into primary tumors plus XRT plus dual-agent immunotherapy on secondary tumor-infiltrating T cells in a mouse model of lung cancer. The hypothesis is that intratumoral primary tumor treatment with NBTXR3 nanoparticle enriches conserved T cell receptor repertoires among metastatic tumor-infiltrating lymphocytes.Four groups of 4 mice each were inoculated subcutaneously with anti-PD1-resistant 344SQR murine lung cancer cells in each hind leg, 3 days apart, to establish 'primary' (right) and 'secondary' (left, which represented metastasis) tumors. All mice received intraperitoneal anti-PD1 and anti-CTLA-4 (IT) on days 4, 7, 10, and 13, and 12-Gy high-dose XRT (HD) to the primary tumors on days 7, 8 and 9. Primary tumors in groups 2 and 4 also received intratumoral NBTXR3 on day 6. Secondary tumors in groups 3 and 4 were irradiated with 1-Gy low-dose XRT (LD) on days 12 and 13. Treatment groups were designated as 1 = HD+IT, 2 = NBTXR3+HD+IT, 3 = HD+LD+IT, and 4 = NBTXR3+HD+LD+IT. All secondary tumors were harvested on day 19 for tumor-infiltrating T-cell RNA extraction and sequencing on an Illumina MiSeq. T-cell receptor (TCR) beta complementarity-determining region 3 (CDR3b) repertoires were analyzed with MiXCR v3.0.12 software, and CDR3b analyses and Circos plots were generated using software. P < 0.05 from Mann-Whitney U tests were considered statistically significant.NBTXR3 significantly increased the proportion of conserved CDR3b clonotypes within the secondary tumors among mice that received only HD XRT to the primary tumors (group 2 vs 1, P = 0.0476). When the secondary tumors were exposed to LD XRT, NBTXR3 still increased the frequency of overlapping clonotypes although not significantly (group 4 vs 3, P = 0.3095). Most interestingly, there were significantly more conserved CDR3b repertoires from the secondary tumors that were shared between groups 2 and 4 that received NBTXR3 compared to those shared between groups 1 and 3 (P < 0.0001). Comparisons of CDR3b normalized Shannon clonality indices and Circos plots did not reveal any difference in repertoire diversity between NBTXR3-treated and non-NBTXR3-treated groups.NBTXR3 radioenhancer nanoparticle, given with immunoradiation, might reshape the TCR repertoire of metastatic lung cancer tumor-infiltrating T cells by driving the selection of conserved CDR3b clonotypes.

DDEL-15. NANOTHERAPEUTIC TARGETING OF TUMOR ENDOTHELIUM FOR ENHANCING DRUG DELIVERY PAST THE BLOOD-BRAIN BARRIER

OBJECTIVEThe Sonic Hedgehog (SHH) medulloblastoma subgroup accounts for ~25% of all cases and has an intermediate prognosis. Current therapies result in devastating morbidities including intellectual disability and secondary malignancies. Although molecularly targeted agents against the SHH pathway have demonstrated efficacy, on-target bone toxicities suggest new therapeutic approaches are needed.METHODSWe investigated the SHH pathway inhibitor, vismodegib, packaged in a fucoidan-based nanoparticle (Fi-Vis) that targets P-selectin expressed on endothelial cells and induced by low-dose ionizing radiation (XRT) in a time- and dose-dependent manner. This P-selectin targeting nanoparticle shows selectivity toward tumor and not normal brain vasculature in a GEM SHH medulloblastoma model as assessed by ex vivo infrared imaging and molecular studies.RESULTSQuantitative RT-PCR analysis of SHH medulloblastoma following single dose XRT and Fi-Vis treatment (10mg/kg) showed synergistic reduction of Gli1 expression (>90% target inhibition). We demonstrate that low-dose XRT (0.25Gy) can induce P-selectin expression specifically on medulloblastoma tumor endothelium and synergize with low-dose Fi-Vis (10mg/kg) to significantly enhance mouse survival (p<0.01) compared to radiation or Fi-Vis alone. Assessment of bone toxicity using micro-CT and histological analysis following Fi-Vis administration in postnatal (P10) mice shows no bone toxicity when compared to free vismodegib. Finally, in vitro studies using bEnd.3 brain endothelial cells and in vivo studies using Cav1 knockout mice suggest a caveolin-1 mediated transcytosis mechanism for nanoparticle entry across the blood-brain barrier.CONCLUSIONSThese data suggest applicability of combined XRT and tumor vasculature-targeted nanotherapeutic dose de-escalation strategies for SHH medulloblastoma with implications for other pediatric brain tumors.

Low-dose radiation treatment enhances systemic antitumor immune responses by overcoming the inhibitory stroma

BackgroundDespite some successes with checkpoint inhibitors for treating cancer, most patients remain refractory to treatment, possibly due to the inhibitory nature of the tumor stroma that impedes the function and...

SCIDOT-41. NANOTHERAPEUTIC TARGETING OF TUMOR ENDOTHELIUM FOR ENHANCING DRUG DELIVERY PAST THE BLOOD-BRAIN BARRIER

Abstract OBJECTIVE The Sonic Hedgehog (SHH) medulloblastoma subgroup accounts for ~25% of all cases and has an intermediate prognosis. Current conventional therapies result in devastating morbidities including intellectual disability and secondary malignancies. Although molecularly targeted agents that inhibit the SHH pathway have demonstrated clinical efficacy, recent studies have shown on-target secondary toxicities on bone development suggesting new therapeutic approaches are needed. METHODS We investigated the efficacy of the SHH pathway inhibitor, Vismodegib packaged in a fucoidan-based nanoparticle (Fi-Vis) that targets P-selectin, a protein overexpressed on vascular endothelial cells and induced by low-dose ionizing radiation (XRT) in a time- and dose-dependent manner. This P-selectin targeting nanoparticle drug delivery system shows selectivity toward tumor vasculature and not normal brain vasculature in a genetic SHH medulloblastoma mouse model as assessed by ex vivo infrared imaging and two-photon intravital imaging. RESULTS Quantitative RT-PCR analysis of SHH medulloblastoma tissue following single dose XRT and Fi-Vis treatment (10mg/kg) showed a synergistic inhibition of Gli1 expression (&gt;90% target inhibition). Furthermore, we demonstrate that very low dose XRT (0.25Gy) can induce P-selectin expression specifically within MB tumor vasculature and synergize with low dose Fi-Vis (10mg/kg) to significantly enhance mouse survival (p&lt;0.01) when compared to radiation or Fi-Vis alone. Furthermore, assessment of bone toxicity using micro-CT and histological analysis following Fi-Vis administration in postnatal (P10) mice shows no bone toxicity when compared to free Vismodegib. Finally, in vitro studies using mouse brain endothelial cells suggest at least in part a caveolin-1 mediated transcytosis mechanism of crossing the endothelial blood-brain barrier. CONCLUSIONS These data suggest applicability of combined XRT and tumor vasculature-targeted nanotherapeutic dose de-escalation strategies for SHH medulloblastoma with implications for other pediatric and adult brain tumors.

Low dose radiotherapy promotes immune-mediated anti-tumor responses

Abstract In an immune competent host, immune surveillance maintains the guards against abnormal cell growth, therefore establishing the need for tumors to evade immunity to grow. Tumors tend to mask themselves with an inhibitory stroma, rich with T regulatory cells, Myeloid-derived suppressor cells, Cancer associated fibroblasts, and pro-tumor M2 macrophages. Despite current advances with checkpoint inhibitors and cell-based therapies, the majority of cancer patients remain refractory to treatment due to the presence of the stroma and the inability of effector cells to penetrate. Radiotherapy (XRT) has been traditionally used to control tumors locally. More recently, we developed the RadScopal technique that combines high dose XRT (H-XRT) to release antigens and prime T-cells with low dose XRT (L-XRT) to overcome the stroma, favor the polarization of M1 macrophages, reduce TGF-β levels, and enhance NK cell infiltration. In the 129Sv/Ev murine model of bilaterally transplanted tumors, RadScopal treatment significantly improved the outcomes of anti-CTLA-4 and anti-PD1 checkpoint inhibitors and controlled the growth of primary as well as distal secondary tumors treated with L-XRT. The RadScopal efficacy was nulled when using nude mice lacking effector adaptive immunity. Moreover, specific immune-cell depletion studies highlighted the importance of CD4+ T-cells and NK cells to carry out the anti-tumor functions. Our radio-immunotherapeutic approach was operative in other models such as Lewis Lung Carcinoma, where L-XRT retarded the growth of secondary tumors. In conclusion, L-XRT significantly augmented checkpoint blockers with potential future application to cell therapies (CAR-T and TCR) to extend the benefits to more patients.

Mesenchymal stem cells selectively engraft into tumor stroma and produce potent antitumor proteins in situ

3025 Background: The formation of stroma is essential for tumor growth and involves complex interactions between malignant cells and non-tumor stromal cells. We have previously demonstrated that IV injected bone marrow derived mesenchymal stem cells (MSC) integrate into solid tumors as stromal elements (Cancer Res 62:3603, 2002; JNCI 96:1593, 2004). Methods: MSC were labeled by a fiber modified Ad vector expressing firefly luciferase (AdLux-F/RGD), injected into normal or tumor-bearing SCID mice, and biodistributed MSC-Lux were imaged utilizing the Xenogen IVIS system. Results: After IP injection, no hMSC-LUX were detected in normal animals after 7 days, while strong punctate regions of LUX-activity were observed in ovarian tumors. Tumor cells transduced with renilla luciferase constructs co- localized with firefly luciferase MSC. Next, we examined whether hMSC-producing interferon-beta (IFNβ-MSC) could inhibit the growth of metastatic tumors in the lungs of SCID mice. When injected IV (4 doses of 106 MSC/week) into SCID mice with pulmonary metastases of carcinomas or melanomas, tumor growth was significantly inhibited as compared with untreated or vector-control MSC controls (p= 0.007). IV injected IFNb-MSC prolonged the survival of mice bearing metastatic breast carcinomas (p=0.001). In an orthotopic, chemoresistant breast cancer model in syngeneic immunocompetent mice, MSC producing IFN-β completely abrograted tumor growth. Localized low-dose XRT to tumors significantly increased the number of tumor-resident MSC. Conclusions: The data suggest that systemically administered gene-modified MSC selectively engraft into the tumor microenvironment and remain resident as part of the tumor architecture. MSC-expressing IFN-β inhibit the growth of melanomas, gliomas, metastatic breast and ovarian cancers in vivo and prolong the survival of mice bearing established tumors. Clinical trials are in preparation. No significant financial relationships to disclose.

Treatment of cholangiocarcinoma with oncolytic herpes simplex virus combined with external beam radiation therapy

Replication-competent oncolytic herpes simplex viruses (HSV), modified by deletion of certain viral growth genes, can selectively target malignant cells. The viral growth gene gamma(1)34.5 has significant homology to GADD34 (growth arrest and DNA damage protein 34), which promotes cell cycle arrest and DNA repair in response to stressors such as radiation (XRT). By upregulating GADD34, XRT may result in greater oncolytic activity of HSV strains deficient in the gamma(1)34.5 gene. The human cholangiocarcinoma cell lines KMBC, SK-ChA-1 and YoMi were treated with NV1023, an oncolytic HSV lacking one copy of gamma(1)34.5. Viral proliferation assays were performed at a multiplicity of infection (MOI, number of viral particles per tumor cell) equal to 1, either alone or after XRT at 250 or 500 cGy. Viral replication was assessed by plaque assay. In vitro cytotoxicity assays were performed using virus at MOIs of 0.01 and 0.1, with or without XRT at 250 cGy and cell survival determined with lactate dehydrogenase assay. Established flank tumors in athymic mice were treated with a single intratumoral injection of virus (10(3) or 10(4) plaque forming units), either alone or after a single dose of XRT at 500 cGy, and tumor volumes measured. RT-PCR was used to measure GADD34 mRNA levels in all cell lines after a single dose of XRT at 250 or 500 cGy. NV1023 was tumoricidal in all three cell lines, but sensitivity to the virus varied. XRT enhanced viral replication in vitro in all cell lines. Combination treatment with low-dose XRT and virus was highly tumoricidal, both in vitro and in vivo. The greatest tumor volume reduction with combination therapy was seen with YoMi cells, the only cell line with increased GADD34 expression after XRT and the only cell line in which a synergistic treatment effect was suggested. In KMBC and SK-ChA-1 cells, neither of which showed increased GADD34 expression after XRT, tumor volume reduction was less pronounced and there was no suggestion of a synergistic effect in either case. Oncolytic HSV are effective in treating human cholangiocarcinoma cell lines, although sensitivity to virus varies. XRT-enhanced viral replication occurs through a mechanism that is not necessarily dependent on GADD34 upregulation. However, XRT-induced upregulation of GADD34 further promotes tumoricidal activity in viral strains deficient in the gamma(1)34.5 gene, resulting in treatment synergy; this effect is cell type dependent. Combined XRT and oncolytic viral therapy is a potentially important treatment strategy that may enhance the therapeutic ratios of both individual therapies.

743 Preoperative radiation dose escalation ± chemotherapy for advanced rectal cancer

Purpose To assess the influence of preoperative radiation does escalation ± concurrent chemotherapy in patients with advanced rectal cancer. Methods Patients with clinical T3/T4 rectal cancer received preoperative XRT ± chemotherapy, followed by surgery. 74 patients received 45 Gy XRT (low-dose group). 82 patients received 55.8 Gy XRT (high-dose group). 33 patients received 55.8 Gy XRT with concurrent 5-FU chemotherapy (CRT group). The 3 groups were compared with respect to post-radiation pathologic stage, local tumor control (LC), disease-specific survival (DSS), freedom from distant metastasis (FDM), and acute toxicity. Results The high-dose XRT and CRT groups had significantly fewer pT3/4 tumors relative to the low-dose XRT group (53% and 51%vs. 70%, respectively, P 0.005, x2) Conclusions The addition of 5-FU chemotherapy to preoperative XRT results in greater downstaging of clinically fixed tumors than XRT alone. The acute toxicity of CRT is greater than that of XRT alone. Patients with clinically fixed rectal cancer benefit most from preoperative CRT.

Adenoid cystic carcinoma of the salivary glands. Management of recurrent, advanced, or persistent disease with hyperthermia and radiation therapy

Adenoid cystic carcinomas (ACC) of the salivary glands are aggressive tumors characterized by multiple late local recurrences and distant metastases. Current therapy includes wide local excision and high-dose postoperative radiation therapy (XRT) (5400 to 7000 cGy). Despite early aggressive treatment, local recurrence remains a major problem with limited safe and effective therapeutic options available. The excellent local responses obtained in four patients (six sites) with ACC of the head and neck treated either with additional low-dose irradiation (2160 to 3420 cGy) in conjunction with two to five hyperthermia (HT) treatments or with full dose XRT and HT as part of the overall treatment plan are reported. All HT treatments were for 45 minutes once steady state conditions were obtained. Monitored intratumoral temperatures for all treatments achieved average maximum (Tmax), average mean (Tave), and average minimum (Tmin) temperatures of 44.2 degrees C, 41.2 degrees C, and 38.9 degrees C, respectively. A complete response was obtained for all six fields with no significant long-term complications. Two patients remain alive and free of local disease at 42 and 63 months of follow-up. Two patients died--one with metastases (with persistent local control) and one with a local recurrence at 9 and 30 months, respectively, after XRT and HT. This is the first report of HT and low-dose XRT in the management of previously irradiated ACC and suggests a potential role for the use of this modality in the treatment of ACC.