Radiation-resistant Tumors Research Articles

Progression of Femoral Osteolytic Metastases after Intramedullary Nailing and Subsequent Salvage Techniques.

Intramedullary nailing insertion from the proximal-to-distal femur is frequently performed for impending and complete pathological femur fractures due to osteolytic metastases. After nailing through cancer-laden bone, residual chemotherapy- and/or radiation-resistant tumor may progress. Progression of osteolysis risks future nail failure or pathological fractures. This study assesses the incidence of cancer progression following intramedullary nailing in a femur-only cohort and describes a percutaneous rod-retaining salvage technique. A single-institution, retrospective study was conducted to identify adult patients who underwent intramedullary nailing for femoral osteolytic lesions for complete or impending nail failure from 2016 to 2023. Progression was defined as enlargement of the pre-existing lesion and/or appearance of new lesions on radiographs. Surgical outcomes were assessed with a combined pain and functional score. A total of 113 patients (median age 66.8 years (IQR = 16.4); median follow-up 6.0 months (IQR = 14.5)) underwent intramedullary nailing. Sixteen patients (14.2%) exhibited post-nailing cancer progression. Pre- and postoperative radiation and chemotherapy did not decrease the odds of cancer progression. Three patients underwent initial open surgical salvage consisting of proximal femur replacement arthroplasty, and six patients did not receive salvage due to poor surgical candidacy or patient choice. Seven patients (median follow-up 10.7 months (IQR = 12.9)) received percutaneous salvage. In this group, pain and functional scores improved by 4.0 points (p = 0.0078) at two-week postoperative follow-up and 2.0 points (p = 0.0312) at the most recent follow-up (mean follow-up 13.0 ± 9.4 months). All three nonambulatory patients became ambulatory, and six patients were able to ambulate independently without walking aids. No major complications were reported 30 days postoperatively. Progression of femoral osteolytic metastases may occur following intramedullary nailing. Continued monitoring of the entire femur is needed to maintain improved functional status and to prevent catastrophic progression of pre-existing lesions or appearance of new lesions. In patients with more proximal metastases only, the customary practice of bringing a long nail from the proximal femur to distal metaphysis should be reconsidered. Furthermore, there is concern of mechanical transport of cancer cells during guide wire insertion, reaming, and rod insertion through cancer laden bone to cancer free distal bone.

Abstract PO3-07-10: Advanced CD8 ImmunoPET imaging predicts radiation response in primary TNBC

Abstract Background: Tumor immunology and immunosuppression has been observed to drive changes in intratumoral response to cytotoxic and immunotherapy in triple negative breast cancer (TNBC). While radiation therapy is standard of care for TNBC, there is a lack of research into the relationship between long term treatment response and CD8 immune infiltration in the context of a radiation resistant model. Positron emission tomography (PET) imaging allows for noninvasive monitoring of the tumor microenvironment that can precede changes in tumor volume, including approaches that allow for immune imaging of CD8 T-cell trafficking. Noninvasive PET imaging of radiation response has the potential to identify windows of enhanced response to secondary therapy and can guide interventional therapy. The goal of this study is to understand how radiation can prime the tumor microenvironment to be combined with other therapeutics in TNBC through changes in CD8 immune infiltration with [89Zr]-CD8 ImmunoPET imaging. Methods: Syngeneic parental 4T1 (radiation sensitive) and a developed radiation-resistant 4T1 sub-cell line were orthotopically injected into BALB/c mice (N = 5 control, N = 9-10 radiation treated for each model). After reaching a tumor volume of ~100 mm3, mice began treatment with 2 Gy fractionated radiation daily from day 0-5. On day 6, mice were injected with ~50 µCi of [89Zr]-CD8 minibody (IAB42) and imaged 24 hours post injection. The mean standardized uptake value (SUV) was quantified and normalized to heart SUV to extract out a tumor:heart SUVratio that describe the CD8 infiltration within tumors. Following imaging, tumors were either excised immediately for immunofluorescence against CD8 or monitored for longitudinal changes in tumor volume. A non-parametric T-test was used to assess for significance between groups. Results: In radiation sensitive 4T1 tumors, [89Zr]-CD8 ImmunoPET imaging indicated a 23% increase in CD8 immune infiltration, in radiation-treated tumors compared to control tumors on day 7 (p = 0.02). These immune alterations occurred prior to any changes in tumor volume (p = 0.63). Longitudinally, radiation treated tumors exhibited significant changes in tumor volume beginning on day 20 (p = 0.02), which is sustained until study endpoint on day 41 (p < 0.01). In radiation resistant 4T1 tumors, no significant change was observed in short term CD8 immune infiltration (p = 0.43) or in longitudinal tumor volume (p = 0.31) in response to radiation therapy, when compared to untreated tumors. Conclusions: [89Zr]-CD8 ImmunoPET imaging reveals significant increases in CD8 immune infiltration that is predictive of eventual response to radiation therapy in radiation sensitive models of TNBC. [89Zr]-CD8 PET imaging of radiation therapy response has the potential to increase the efficacy of precision medicine and prime the tumor microenvironment for secondary therapeutics, thereby improving tumor kill and reducing patient toxicity. Citation Format: Patrick Song, Shannon Lynch, Chloe DeMellier, Alessandro Mascioni, Fang Jia, Anna Sorace. Advanced CD8 ImmunoPET imaging predicts radiation response in primary TNBC [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-07-10.

Abstract 1120: CD8 ImmunoPET imaging identifies an immunogenically active tumor microenvironment following radiotherapy in primary TNBC

Abstract Background: Tumor immune composition has been shown to drive therapy response in triple negative breast cancer (TNBC). Radiation is a component of standard-of-care treatments for TNBC, however immune populations have not been studied in the context of radiation resistance in breast cancer. Positron emission tomography (PET) allows for quantifying molecular signatures of the tumor microenvironment, which can precede anatomical changes in tumor volume. Recently, development of immune-targeted imaging approaches has allowed for noninvasive quantification of immune cells, allowing for quantification and long-term therapy response. Preclinical studies have demonstrated that combining therapies, such as radiotherapy and immunotherapy, could be beneficial in treating cancer. The goal of this study is to quantify changes in immune infiltration with [89Zr]-CD8 ImmunoPET following radiation therapy to determine variations in immune activation that can affect therapeutic response in radiation sensitive or radiation resistant tumors. Methods: Syngeneic radiation sensitive 4T1 or a radiation-resistant 4T1 tumors were treated with fractionated radiation (2 Gy/day) from day 0-5, injected with [89Zr]-CD8 minibody on day 6, and imaged with [89Zr]-CD8 PET on day 7. Following imaging, tumors were excised for biological assays (N = 20) or monitored for longitudinal changes in tumor volume (N = 15). Biological assays included CD8 immunofluorescence or assessment of immune population differences with flow cytometry against T-cells (CD3, CD4, CD8, IFN-gamma), macrophages (CD45, F4/80, CD86 and CD206), and natural killer cells (CD56 and CD16). A non-parametric T-test was used to assess for statistical differences. Results: In radiation sensitive tumors, [89Zr]-CD8 PET indicated that radiation significantly increases CD8 immune infiltration (p = 0.02) compared to controls, which was correlated with immunofluorescence (p < 0.01). Immune infiltration was increased in radiation sensitive tumors when treated with radiation therapy (4.6-fold increase in CD86+ M1-like macrophages, 4.7-fold increase in CD206+ M2-like macrophages, 5.3-fold increase in CD8 T-cells and 4.3-fold increase in CD4 T-cells) compared to control tumors. Radiation was observed to increase cytotoxic CD8 immune infiltration in radiation sensitive tumors. In radiation resistant tumors, no significant difference was observed in either CD8 immune infiltration or tumor volume in treated tumors compared to controls (p = 0.63). Conclusions: [89Zr]-CD8 ImmunoPET reveals significant increases in CD8 immune infiltration in a radiation sensitive model of TNBC and identifies an immune altered tumor immune microenvironment. Fractionated radiotherapy was also observed to significantly increase the infiltration of innate and adaptive immune cells. Citation Format: Patrick N. Song, Shannon E. Lynch, Chloe T. DeMellier, Alessandro Mascioni, Jia Fang, Anna G. Sorace. CD8 ImmunoPET imaging identifies an immunogenically active tumor microenvironment following radiotherapy in primary TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1120.

Imaging Role in Diagnosis, Prognosis, and Treatment Response Prediction Associated with High-grade Glioma.

Glioma is one of the most drug and radiation-resistant tumors. Gliomas suffer from inter- and intratumor heterogeneity which makes the outcome of similar treatment protocols vary from patient to patient. This article is aimed to overview the potential imaging markers for individual diagnosis, prognosis, and treatment response prediction in malignant glioma. Furthermore, the correlation between imaging findings and biological and clinical information of glioma patients is reviewed. The search strategy in this study is to select related studies from scientific websites such as PubMed, Scopus, Google Scholar, and Web of Science published until 2022. It comprised a combination of keywords such as Biomarkers, Diagnosis, Prognosis, Imaging techniques, and malignant glioma, according to Medical Subject Headings. Some imaging parameters that are effective in glioma management include: ADC, FA, Ktrans, regional cerebral blood volume (rCBV), cerebral blood flow (CBF), ve, Cho/NAA and lactate/lipid ratios, intratumoral uptake of 18F-FET (for diagnostic application), RD, ADC, ve, vp, Ktrans, CBFT1, rCBV, tumor blood flow, Cho/NAA, lactate/lipid, MI/Cho, uptakes of 18F-FET, 11C-MET, and 18F-FLT (for prognostic and predictive application). Cerebral blood volume and Ktrans are related to molecular markers such as vascular endothelial growth factor (VEGF). Preoperative ADCmin value of GBM tumors is associated with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status. 2-hydroxyglutarate metabolite and dynamic 18F-FDOPA positron emission tomography uptake are related to isocitrate dehydrogenase (IDH) mutations. Parameters including ADC, RD, FA, rCBV, Ktrans, vp, and uptake of 18F-FET are useful for diagnosis, prognosis, and treatment response prediction in glioma. A significant correlation between molecular markers such as VEGF, MGMT, and IDH mutations with some diffusion and perfusion imaging parameters has been identified.

Abstract C119: Combining forces: Integrating engineered bacterial therapeutics with targeted therapies for advanced-stage non-small cell lung cancer

Abstract Currently there are about 400 single-agent targeted therapies in various stages of clinical trials for non-small cell lung cancer (NSCLC) worldwide. However, less than 10% of them have been utilized in combination treatment to improve efficacy of advanced-stage NSCLC due to safety issues. As a result, emerging and safe treatment modalities such as engineered bacteria therapy, commonly known as “bugs as drugs”, in ongoing clinical trials (NCT00004988, NCT04589234, NCT05038150) for advanced-NSCLCs - have not been combined with current standards of care. We hypothesized that If advanced-stage NSCLC tumors are dependent on altered growth signaling upon single-agent bacteria therapy, blocking those signaling with current targeted therapy may provide a promising combination treatment strategy to achieve better efficacy. To test this, we first treated NSCLC-derived spheroids with engineered bacteria producing Theta toxin after localizing in the hypoxic core and eliminating spheroids from inside. Second, we characterized the signaling pathways of bacteria-treated NSCLC spheroids by RNA-seq followed by Gene Set Enrichment Analysis. Third, we blocked altered signaling with current targeted therapies to achieve better efficacy. Finally, we designed an optimum combination treatment and validated it in vivo in xenografted NSCLCs. We found that bacterially secreted Theta toxin significantly altered PI3K/AKT/mTOR signaling, cell cycle checkpoint and DNA repair pathways (normalized p-value < 0.5 and falls discovery rate q-value < 0.5) in multiple NSCLC spheroids, including one established from a chemotherapy and radiation-resistant tumor. We chose current targeted inhibitors of AKT (MK2206), CHK (AZD7762), WNT (IWR-1), ERK (SCH772984), MEK (Selumetinib), and 2 chemotherapy drugs (paclitaxel, pemetrexed) to treat these bacterially-treated NSCLC spheroids. We found that blocking AKT or CHK signaling in combination with bacteria therapy significantly decreased (p<0.002) NSCLC spheroid viability. Honing in on AKT signaling, we validated bacterially secreted Theta toxin induced AKT phosphorylation in NSCLC cells in vitro that we were able to block with MK2206 treatment. Finally, we tested this combination of bacterially secreted Theta toxin and MK2206 in xenografted NSCLC tumors. The combination treatment significantly improved efficacy in all tumors (p<0.001) compared to single-agent therapy (either engineered bacteria or MK2206 alone) without any additional toxicity suggesting a promising translational treatment strategy. We conclude that our modular experimental and analytical pipeline can be exploited to characterize engineered bacteria therapies and combine them with specific targeted therapy to expedite the translation of an untapped area of drug development with biological therapeutic agents for advanced-stage NSCLC. Citation Format: Dhruba Deb, Tal Danino. Combining forces: Integrating engineered bacterial therapeutics with targeted therapies for advanced-stage non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C119.

An international approach to estimating the indications and number of eligible patients for carbon ion radiation therapy (CIRT) in Australia

Background and purposeTo establish the treatment indications and potential patient numbers for carbon ion radiation therapy (CIRT) at the proposed national carbon ion (and proton) therapy facility in the Westmead precinct, New South Wales (NSW), Australia. MethodsAn expert panel was convened, including representatives of four operational and two proposed international carbon ion facilities, as well as NSW-based CIRT stakeholders. They met virtually to consider CIRT available evidence and experience. Information regarding Japanese CIRT was provided pre- and post- the virtual meeting. Published information for South Korea was included in discussions. ResultsThere was jurisdictional variation in the tumours treated by CIRT due to differing incidences of some tumours, referral patterns, differences in decisions regarding which tumours to prioritise, CIRT resources available and funding arrangements. The greatest level of consensus was reached that CIRT in Australia can be justified currently for patients with adenoid cystic carcinomas and mucosal melanomas of the head and neck, hepatocellular cancer and liver metastases, base of skull meningiomas, chordomas and chondrosarcomas. Almost 1400 Australian patients annually meet the consensus-derived indications now. ConclusionA conservative estimate is that 1% of cancer patients in Australia (or 2% of patients recommended for radiation therapy) may preferentially benefit from CIRT for initial therapy of radiation resistant tumours, or to boost persistently active disease after other therapies, or for re-irradiation of recurrent disease. On this basis, one national carbon ion facility with up to four treatment rooms is justified for Australian patients.

Predicting tumor repopulation through the gene panel derived from radiation resistant colorectal cancer cells

BackgroundTumor cells with the capability of radiation resistance can escape the fate of cell death after radiotherapy, serving as the main cause of treatment failure. Repopulation of tumors after radiotherapy is dominated by this group of residual cells, which greatly reduce the sensitivity of recurrent tumors to the therapy, resulting in poor clinical outcomes. Therefore, revealing the mechanism of radiation resistant cells participating in tumor repopulation is of vital importance for cancer patients to obtain a better prognosis.MethodsCo-expressed genes were searched by using genetic data of radiation resistant cells (from GEO database) and TCGA colorectal cancer. Univariate and multivariate Cox regression analysis were performed to define the most significant co-expressed genes for establishing prognostic indicator. Logistic analysis, WGCNA analysis, and other types of tumors were included to verify the predictive ability of the indicator. RT-qPCR was carried out to test expression level of key genes in colorectal cancer cell lines. Colongenic assay was utilized to test the radio-sensitivity and repopulation ability of key gene knockdown cells.ResultsPrognostic indicator based on TCGA colorectal cancer patients containing four key radiation resistance genes (LGR5, KCNN4, TNS4, CENPH) was established. The indicator was shown to be significantly correlated with the prognosis of colorectal cancer patients undergoing radiotherapy, and also had an acceptable predictive effect in the other five types of cancer. RT-qPCR showed that expression level of key genes was basically consistent with the radiation resistance level of colorectal cancer cells. The clonogenic ability of all key gene knockdown cells decreased after radiation treatment compared with the control groups.ConclusionsOur data suggest that LGR5, KCNN4, TNS4 and CENPH are correlated with radiation sensitivity of colorectal cancer cells, and the indicator composed by them can reflect the prognosis of colorectal cancer patients undergoing radiation therapy. Our data provide an evidence of radiation resistant tumor cells involved in tumor repopulation, and give patients undergoing radiotherapy an approving prognostic indicator with regard to tumor progression.

Abstract 1088: Acquired radiation resistance induces thiol-dependent cisplatin cross-resistance

Abstract Purpose. Resistance to radiation, both intrinsic and acquired, remains a significant clinical challenge. It is therefore import to identify the underlying molecular and cellular features involved in this resistance. Materials and Methods. We generated genetically-matched counterparts of various degrees of radiation sensitivity in non-small cell lung carcinoma (NSCLC) tumor models to define characteristics of acquired resistance through repeated exposures to conventional X-ray radiation. Results. As assessed by cell viability and clonogenic assays murine Lewis lung carcinoma (LLC) and human A549 cell lines acquired an approximate 1.5 - 2-fold increase in radiation resistance (RR) as compared to its parental counterpart. Morphologically, we found that A549-RR exhibited a greater nucleus-to-cytosol (N/C) ratio as compared to its respective counterpart. Since the N/C ratio has been linked to the differentiation state, we next investigated the epithelial-to-mesenchymal transition (EMT) phenotype and cellular plasticity. We found that A549 have a greater radiation-induced plasticity, as measured by E-cadherin, vimentin and double positive (DP) modulation, as compared to LLC. Additionally, migration was suppressed in A549-RR, as compared to A549. Subsequently, we confirmed that the LLC-RR and A549-RR are also more resistant to radiotherapy than their isogenic-matched counterpart in vivo. Remarkably, we found that the acquired radiation resistance also induced resistance to first-line chemotherapeutic cisplatin, but not carboplatin or oxaliplatin. This cross-resistance was attributed to induced elevated thiol levels. Decreasing the amount of thiols with buthionine sulfoximine (BSO), a gamma-glutamylcysteine synthetase inhibitor, sensitized the resistant cells to cisplatin, to levels similar as parental cells. Conclusions. By generating genetically-matched radiation resistant NSCLC tumor models we were able to identify and overcome cisplatin cross-resistance. This is an important finding arguing for multi-modality treatment regimens with the potential of improving clinical outcomes in the future. Citation Format: Samir V. Jenkins, Shruti Shah, Azemat Jamshidi-Parsian, Amir Mortazavi, Gunnar Boysen, Kieng B. Vang, Robert J. Griffin, Narasimhan Rajaram, Ruud P. Dings. Acquired radiation resistance induces thiol-dependent cisplatin cross-resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1088.

Functionalized Hybrid Iron Oxide–Gold Nanoparticles Targeting Membrane Hsp70 Radiosensitize Triple-Negative Breast Cancer Cells by ROS-Mediated Apoptosis

Triple-negative breast cancer (TNBC) a highly aggressive tumor entity with an unfavorable prognosis, is treated by multimodal therapies, including ionizing radiation (IR). Radiation-resistant tumor cells, as well as induced normal tissue toxicity, contribute to the poor clinical outcome of the disease. In this study, we investigated the potential of novel hybrid iron oxide (Fe3O4)-gold (Au) nanoparticles (FeAuNPs) functionalized with the heat shock protein 70 (Hsp70) tumor-penetrating peptide (TPP) and coupled via a PEG4 linker (TPP-PEG4-FeAuNPs) to improve tumor targeting and uptake of NPs and to break radioresistance in TNBC cell lines 4T1 and MDA-MB-231. Hsp70 is overexpressed in the cytosol and abundantly presented on the cell membrane (mHsp70) of highly aggressive tumor cells, including TNBCs, but not on corresponding normal cells, thus providing a tumor-specific target. The Fe3O4 core of the NPs can serve as a contrast agent enabling magnetic resonance imaging (MRI) of the tumor, and the nanogold shell radiosensitizes tumor cells by the release of secondary electrons (Auger electrons) upon X-ray irradiation. We demonstrated that the accumulation of TPP-PEG4-FeAuNPs into mHsp70-positive TNBC cells was superior to that of non-conjugated FeAuNPs and FeAuNPs functionalized with a non-specific, scrambled peptide (NGL). After a 24 h co-incubation period of 4T1 and MDA-MB-231 cells with TPP-PEG4-FeAuNPs, but not with control hybrid NPs, ionizing irradiation (IR) causes a cell cycle arrest at G2/M and induces DNA double-strand breaks, thus triggering apoptotic cell death. Since the radiosensitizing effect was completely abolished in the presence of the ROS inhibitor N-acetyl-L-cysteine (NAC), we assume that the TPP-PEG4-FeAuNP-induced apoptosis is mediated via an increased production of ROS.

MODL-31. Novel in vivo models of post-radiation recurrent pediatric high-grade glioma show increased susceptibility to MAPK inhibition

BACKGROUND: Pediatric high-grade glioma (pHGG) is the most common cause of childhood cancer mortality, with median survival of less than one year. The standard of care for pHGG includes radiation therapy (RT), but almost all patients who respond initially relapse with aggressive, radiation-resistant disease. Matched primary/recurrent models and effective therapies for recurrent pHGG have not been adequately developed. METHODS: Orthotopic patient derived xenograft (PDX) models of radiation-resistant pHGG were developed by implanting BT245 (H3K27M) and HSJD-GBM-001 (GBM1, H3-wt) cells into mice. Resultant tumors were irradiated at 8Gy (4Gy x 2d) and allowed to regrow. Recurrent, radiation-resistant tumors (BT245MR and GBM1MR) and initial tumors (BT245M and GBM1M) were collected for bulk RNA-Seq analysis and ex vivo cell culture. Dose-response experiments were conducted in explant cells to identify drugs with increased effectiveness against recurrent (MR) cell lines versus wild-type cell lines. RESULTS: In both BT245 and GBM1 explant cells, geneset enrichment analysis (GSEA) showed upregulated mesenchymal (BT245MR: NES=3.4, FDR=0; GBM1MR: NES=2.4, FDR<0.002) and hypoxia-related (BT245MR: NES=3.6, FDR=0; GBM1MR: NES=3.4, FDR=0) pathways. GBM1MR tumors also showed enrichment in MAPK signaling (NES=4.7, FDR=0). RNA processing (NES=-6.5, FDR=0) and translation (NES=-5.8, FDR=0) pathways were depleted in BT245MR cells, and DNA repair pathways (NES=-5.8, FDR=0) were depleted in GBM1MR cells. The MAPK inhibitor trametinib demonstrated increased effectiveness in BT245MR and GBM1MR cell lines versus wild-type controls (BT245 IC50 158.7nM (wt) vs. 56.0 (MR), p<0.05; GBM1 IC50 150.7 (wt) vs. 53.4 (MR), p<0.05). BT245MR and GBM1MR cells reliably form PDX tumors after injection into mice. CONCLUSION: BT245MR and GBM1MR represent novel in vivo models of radiation-resistant pHGG, with gene expression alterations consistent with an irradiated phenotype. GSEA and in vitro dose-response data suggest MAPK inhibition may be effective in radiation-resistant pHGG. We are currently conducting in vivo experiments to validate increased trametinib sensitivity in these models.

PEGPH20, a PEGylated human hyaluronidase, induces radiosensitization by reoxygenation in pancreatic cancer xenografts. A molecular imaging study

PurposePEGylated human hyaluronidase (PEGPH20) enzymatically depletes hyaluronan, an important component of the extracellular matrix, increasing the delivery of therapeutic molecules. Combinations of chemotherapy and PEGPH20, however, have been unsuccessful in Phase III clinical trials. We hypothesize that by increasing tumor oxygenation by improving vascular patency and perfusion, PEGPH20 will also act as a radiosensitization agent. Experimental DesignThe effect of PEGPH20 on radiation treatment was analyzed with respect to tumor growth, survival time, p02, local blood volume, and the perfusion/permeability of blood vessels in a human pancreatic adenocarcinoma BxPC3 mouse model overexpressing hyaluronan synthase 3 (HAS3). ResultsMice overexpressing HAS3 developed fast growing, radiation resistant tumors that became rapidly more hypoxic as time progressed. Treatment with PEGPH20 increased survival times when used in combination with radiation therapy, significantly more than either radiation therapy or PEGPH20 alone. In mice that overexpressed HAS3, EPR imaging showed an increase in local pO2 that could be linked to increases in perfusion/permeability and local blood volume immediately after PEGPH20 treatment. Hyperpolarized [1-13C] pyruvate suggested PEGPH20 caused a metabolic shift towards decreased glycolytic flux. These effects were confined to the mice overexpressing HAS3 - no effect of PEGPH20 on survival, radiation treatment, or pO2 was seen in wild type BxPC3 tumors. ConclusionsPEGPH20 may be useful for radiosensitization of pancreatic cancer but only in the subset of tumors with substantial hyaluronan accumulation. The response of the treatment may potentially be monitored by non-invasive imaging of the hemodynamic and metabolic changes in the tumor microenvironment.

18F]Fluoro-DCP, a first generation PET radiotracer for monitoring protein sulfenylation in vivo

Redox metabolism plays essential functions in the pathology of cancer and many other diseases. While several radiotracers for imaging redox metabolism have been developed, there are no reports of radiotracers for in vivo imaging of protein oxidation. Here we take the first step towards this goal and describe the synthesis and kinetic properties of a new positron emission tomography (PET) [18F]Fluoro-DCP radiotracer for in vivo imaging of protein sulfenylation. Time course biodistribution and PET/CT studies using xenograft animal models of Head and Neck Squamous Cell Cancer (HNSCC) demonstrate its capability to distinguish between tumors with radiation sensitive and resistant phenotypes consistent with previous reports of decreased protein sulfenylation in clinical specimens of radiation resistant HNSCC. We envision further development of this technology to aid research efforts towards improving diagnosis of patients with radiation resistant tumors.

Combined Therapy with microRNA-Expressing Salmonella and Irradiation in Melanoma.

Anticancer treatment strategies using bacteria as a vector are currently expanding with the development of anticancer drugs. Here, we present a research strategy to develop anticancer drugs using bacteria that contain miRNAs. We also present a strategy for the development of novel bacterial anticancer drugs in combination with radiation. Salmonella strains expressing miRNA were produced by modifying the miRNA expression vector encoding INHA, a radiation-resistant gene developed previously. The anticancer effect of INHA was confirmed using skin cancer cell lines. We also tested a combination strategy comprising bacteria and radiation for its anticancer efficacy against radiation-resistant mouse melanoma to increase the efficacy of radiation therapy as a novel strategy. The recombinant strain was confirmed to promote effective cell death even when combined with radiation therapy, which exerts its cytotoxicity by enhancing reactive oxygen species production. Moreover, a combination of bacterial and radiation therapy enhanced radiotherapy efficacy. When combined with radiation therapy, bacterial therapy exhibited effective anti-cancer properties even when administered to animals harboring radiation-resistant tumors. This strategy may promote the secretion of cytokines in cells and more effectively reduce the number of bacteria remaining in the animal. Thus, this study may lead to the development of a strategy to improve the effectiveness of radiation therapy using Salmonella expressing cancer-specific miRNA for intractable cancers such as those resistant to radiation.

High-Complexity cellular barcoding and clonal tracing reveals stochastic and deterministic parameters of radiation resistance.

It is elusive whether clonal selection of tumor cells in response to ionizing radiation (IR) is a deterministic or stochastic process. With high resolution clonal barcoding and tracking of over 400 000 HNSCC patient-derived tumor cells the clonal dynamics of tumor cells in response to IR was analyzed. Fractionated IR induced a strong selective pressure for clonal reduction which significantly exceeded uniform clonal survival probabilities indicative for a strong clone-to-clone difference within tumor cell lines. IR induced clonal reduction affected the majority of tumor cells ranging between 96% and 75% and correlated to the degree of radiation sensitivity. Survival to IR is driven by a deterministic clonal selection of a smaller population which commonly survives radiation, while increased clonogenic capacity is a result of clonal competition of cells which have been selected stochastically. A 2-fold increase in radiation resistance results in a 4-fold (P< .05) higher deterministic clonal selection showing that the ratio of these parameters is amenable to radiation sensitivity which correlates to prognostic biomarkers of HNSCC. Evidence for the existence of a rare subpopulation with an intrinsically radiation resistant phenotype commonly surviving IR was found at a frequency of 0.6% to 3.3% (P< .001, FDR 3%). With cellular barcoding we introduce a novel functional heterogeneity associated qualitative readout for tracking dynamics of clonogenic survival in response to radiation. This enables the quantification of intrinsically radiation resistant tumor cells from patient samples and reveals the contribution of stochastic and deterministic clonal selection processes in response to IR.

Studies on Screening of MicroRNA Related to Irradiation Response and the Regulation Mechanism of miRNA-96-5p on Irradiation Resistance in Rectal Cancer Cells

<h3>Purpose/Objective(s)</h3> To screen microRNAs that are positively correlated with radiation resistance in rectal cancer, and to investigate their regulatory mechanism. <h3>Materials/Methods</h3> Fresh rectal cancer tissues were obtained intraoperatively and divided into two equal parts, one of which was pre-stored in a refrigerator at -150℃, and the other was constructed with Patients Derived Xenograft (PDX) models for rectal cancer and irradiated to transplantation tumor screen out radiation-sensitive tumor tissues and radiation-resistant tumor tissues. According to the screening results, the pre-stored samples were screened for differential microRNAs in radiation reactivity, and the microRNAs that were positively correlated with irradiation resistance were obtained. On the base of screening results, the pre-stored samples were screened for differential microRNAs in irradiation reactivity by miRNAs microarrays, and the microRNAs that were positively correlated with irradiation resistance were obtained. Biological prediction software was used to predict the target genes regulated by microRNA, and the prediction results were verified in radiation-resistant rectal cancer cell lines. Lentivirus plasmid transfection technique was used to construct rectal cancer cell lines with down-regulated target microRNA expression, in order to further verify the effects of the changes in the expression of the microRNA on the radiation reactivity, proliferation, invasion and migration of rectal cancer cells. The target genes regulated by microRNAs were screened and verified, and the expression of the key genes in the signal transduction pathways involved in the regulation were detected. <h3>Results</h3> The results of microRNA screening and validation showed that there were 14 microRNAs with fold change > 1.5. Among them, miRNA-552-3p, miRNA-96-5p, miRNA-182-5p, and miRNA-183-5p were up-regulated in irradiation-resistant tumor tissues. The enrichment signaling pathway analysis showed that the Wnt signal transduction pathway was highly active in irradiation-resistant tumor tissues. The clone formation experiment under radiation interference showed that HR-8348 was the most radiation-resistant cell line among the five cell lines, and the expression level of miRNA-96-5p was positively correlated with the radiation-resistant ability of rectal cancer cell lines. The results of prediction and validation showed that the expression of GPC3 gene was directly regulated by miRNA-96-5p, and the down-regulated expression of miRNA-96-5p inhibited the activity of Wnt signal transduction pathway. <h3>Conclusion</h3> The high expression of miRNA-96-5p enhanced the radiation resistance of rectal cancer cells, and its effect may be related to the down-regulation of GPC3 expression and the abnormal activation of Wnt signal transduction pathway.

Prediction of the effects of radiation therapy in esophageal cancer using diffusion and perfusion MRI

Chemoradiation therapy (CRT) of locally advanced esophageal cancer (LAEC), although improving outcomes of patients, still results in 50% of local failure. An early prediction could identify patients at high risk of poor response for individualized adaptive treatment. We aimed to investigate physiological changes in LAEC using diffusion and perfusion magnetic resonance imaging (MRI) for early prediction of treatment response. In the study, 115 LAEC patients treated with CRT were enrolled (67 in the discovery cohort and 48 in the validation cohort). MRI scans were performed before radiotherapy (pre‐RT) and at week 3 during RT (mid‐RT). Gross tumor volume (GTV) of primary tumor was delineated on T2‐weighted images. Within the GTV, the hypercellularity volume (VHC) and high blood volume (VHBV) were defined based on the analysis of ADC and fractional plasma volume (Vp) histogram distributions within the tumors in the discovery cohort. The median GTVs were 28 cc ± 2.2 cc at pre‐RT and 16.7 cc ± 1.5 cc at mid‐RT. Respectively, VHC and VHBV decreased from 4.7 cc ± 0.7 cc and 5.7 cc ± 0.7 cc at pre‐RT to 2.8 cc ± 0.4 cc and 3.5 cc ± 0.5 cc at mid‐RT. Smaller VHC at mid‐RT (area under the curve [AUC] = 0.67, P = .05; AUC = 0.66, P = .05) and further decrease in VHC at mid‐RT (AUC = 0.7, P = .01; AUC = 0.69, P = .03) were associated with longer progression‐free survival (PFS) in both discovery and validation cohort. No significant predictive effects were shown in GTV and VHBV at any time point. In conclusion, we demonstrated that VHC represents aggressive subvolumes in LAEC. Further analysis will be carried out to confirm the correlations between the changes in image‐phenotype subvolumes and local failure to determine the radiation‐resistant tumor subvolumes, which may be useful for dose escalation.

Bone Metastases: State of the Art in Minimally Invasive Interventional Oncology.

Bone is the third most common site involved by cancer metastases, and skeleton-related events such as intractable pain due to direct osseous tumor involvement, pathologic fracture, and neurologic deficits as a consequence of nerve or spinal cord compression often affect patients' functional independence and quality of life unfavorably. The annual medical-economic burden related to bone metastases is a substantial component of the total direct medical cost estimated by the National Institutes of Health. There have been substantial recent advances in percutaneous image-guided minimally invasive musculoskeletal oncologic interventions for the management of patients with osseous metastatic disease. These advances include thermal ablation, cementation with or without osseous reinforcement with implants, osteosynthesis, thermal and chemical neurolyses, and palliative injections, which are progressively incorporated into the management paradigm for such patients. These interventions are performed in conjunction with or are supplemented by adjuvant radiation therapy, systemic therapy, surgery, or analgesic agents to achieve durable pain palliation, local tumor control, or cure, and they provide a robust armamentarium for interventional radiologists to achieve safe and effective treatment in a multidisciplinary setting. In addition, these procedures are shifting the patient management paradigm in modern-era practice. The authors detail the state of the art in minimally invasive percutaneous image-guided musculoskeletal oncologic interventions and the role of radiologists in managing patients with skeletal metastases. ©RSNA, 2021.

Personalized Genome-Scale Metabolic Models Identify Targets of Redox Metabolism in Radiation-Resistant Tumors.

Redox cofactor production is integral toward antioxidant generation, clearance of reactive oxygen species, and overall tumor response to ionizing radiation treatment. To identify systems-level alterations in redox metabolism that confer resistance to radiation therapy, we developed a bioinformatics pipeline for integrating multi-omics data into personalized genome-scale flux balance analysis models of 716 radiation-sensitive and 199 radiation-resistant tumors. These models collectively predicted that radiation-resistant tumors reroute metabolic flux to increase mitochondrial NADPH stores and reactive oxygen species (ROS) scavenging. Simulated genome-wide knockout screens agreed with experimental siRNA gene knockdowns in matched radiation-sensitive and radiation-resistant cancer cell lines, revealing gene targets involved in mitochondrial NADPH production, central carbon metabolism, and folate metabolism that allow for selective inhibition of glutathione production and H2O2 clearance in radiation-resistant cancers. This systems approach represents a significant advancement in developing quantitative genome-scale models of redox metabolism and identifying personalized metabolic targets for improving radiation sensitivity in individual cancer patients.

Spectroscopic investigation of radiation-induced reoxygenation in radiation-resistant tumors

Fractionated radiation therapy is believed to reoxygenate and subsequently radiosensitize surviving hypoxic cancer cells. Measuring tumor reoxygenation between radiation fractions could conceivably provide an early biomarker of treatment response. However, the relationship between tumor reoxygenation and local control is not well understood. We used noninvasive optical fiber-based diffuse reflectance spectroscopy to monitor radiation-induced changes in hemoglobin oxygen saturation (sO2) in tumor xenografts grown from two head and neck squamous cell carcinoma cell lines – UM-SCC-22B and UM-SCC-47. Tumors were treated with 4 doses of 2 Gy over 2 consecutive weeks and diffuse reflectance spectra were acquired every day during the 2-week period. There was a statistically significant increase in sO2 in the treatment-responsive UM-SCC-22B tumors immediately following radiation. This reoxygenation trend was due to an increase in oxygenated hemoglobin (HbO2) and disappeared over the next 48 h as sO2 returned to preradiation baseline values. Conversely, sO2 in the relatively radiation-resistant UM-SCC-47 tumors increased after every dose of radiation and was driven by a significant decrease in deoxygenated hemoglobin (dHb). Immunohistochemical analysis revealed significantly elevated expression of hypoxia-inducible factor (HIF-1) in the UM-SCC-47 tumors prior to radiation and up to 48 h postradiation compared with the UM-SCC-22B tumors. Our observation of a decrease in dHb, a corresponding increase in sO2, as well as greater HIF-1α expression only in UM-SCC-47 tumors strongly suggests that the reoxygenation within these tumors is due to a decrease in oxygen consumption in the cancer cells, which could potentially play a role in promoting radiation resistance.

Abstract 279: Long non-coding RNAs in glioblastoma tumor recurrence and therapy resistance

Abstract Glioblastoma multiforme (GBM) is a devastating disease which invariably recurs and is often resistant to standard therapies. GBM patient-derived xenoline (PDX) models of tumor recurrence and radiation resistance were created using serial in vivo selection against radiation therapy (6 × 2Gy fractions over 2 weeks for 6+ rounds). This produced 8 isogenic pairs of patient matched radiation-sensitive, primary GBM PDX to radiation-resistant, recurrent GBM PDX. Long non-coding RNAs (lncRNA) represent novel regulatory mechanisms for therapy resistance and tumor recurrence in GBM. An in silico informatics pipeline employing differential expression, differential gene correlation, machine learning, and semantic networking was devised to identify coding and non-coding transcripts related to radiation resistance from deep total RNA sequencing. This analysis revealed 269 lncRNA and 947 coding transcripts that are associated with adaptive radiation resistance. LncRNA:RNA and lncRNA:DNA interaction prediction software was employed to further uncover mechanisms of epigenetic regulation by lncRNAs. A subset of the lncRNA transcripts were predicted to interact directly with other coding transcripts while other lncRNAs have predicted interactions with DNA in regulatory regions of the human genome. We are further validating lncRNA:DNA interactions through in silico methods to determine if genes proximal to these interaction sites are related to tumorigenesis or therapy response. Semantic linkages have been predicted from references in the current literature for lncRNAs and key molecular processes such as DNA damage response, extracellular matrix, and chromatin remodeling. Modules of highly correlated genes, including lncRNAs, have been identified that are differentially regulated between radiation sensitive and resistant tumors. Cross-validation between our in silico approaches have confirmed high-confidence associations of a subset of lncRNAs with acquired radiation resistance. Subsequently, we will validate the expression and phenotypic relevance of high-confidence transcripts in our cohort of PDX models derived from recurrent GBM tumors. In conclusion, recurrence of therapy resistant GBM is responsible for patient mortality. Not all patients qualify for surgical resection or for chemotherapy, but radiation is almost a universally tolerated therapy. We have generated a novel model of tumor recurrence and radiation resistance that recapitulates recurrent tumor physiology. We have discovered 269 lncRNAs associated with radiation resistance that suggests potential regulatory mechanisms through nucleic acid binding. Evidence suggests that lncRNAs most likely contribute to acquired resistance through epigenetic regulation of transcription and chromatin state. These transcripts may provide novel, druggable targets for treating therapy resistant, recurrent GBM. Citation Format: Christian Tyler Stackhouse, James R. Rowland, Jelai Wang, Thanh Nguyen, Zongliang Yue, Jake Y. Chen, Lara Ianov, G. Yancey Gillespie, Christopher D. Willey. Long non-coding RNAs in glioblastoma tumor recurrence and therapy resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 279.