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Single-cell analysis clustering for gene profiling.

Purpose:Intratumoral immunosuppression mediated by myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) represents a potential mechanism of immune checkpoint inhibitor (ICI) resistance in solid tumors. By promoting TAM and MDSC infiltration, IL1β may drive adaptive and innate immune resistance in renal cell carcinoma (RCC) and in other tumor types.Experimental Design:Using the RENCA model of RCC, we evaluated clinically relevant combinations of anti-IL1β plus either anti-PD-1 or the multitargeted tyrosine kinase inhibitor (TKI), cabozantinib. We performed comprehensive immune profiling of established RENCA tumors via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA sequencing (RNA-seq). Similar analyses were extended to the MC38 tumor model.Results:Analyses via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA-seq showed that anti-IL1β reduces infiltration of polymorphonuclear MDSCs and TAMs. Combination treatment with anti-IL1β plus anti-PD-1 or cabozantinib showed increased antitumor activity that was associated with decreases in immunosuppressive MDSCs and increases in M1-like TAMs.Conclusions:Single-cell RNA-seq analyses show that IL1β blockade and ICI or TKI remodel the myeloid compartment through nonredundant, relatively T-cell–independent mechanisms. IL1β is an upstream mediator of adaptive myeloid resistance and represents a potential target for kidney cancer immunotherapy.

Immune cell phenotyping of tumor draining lymph nodes from RENCA tumor bearing mice.

Visualization of top 5 upregulated RNA transcripts associated with each cluster.

Clusters from Intratumoral Immunocytes by Spectral Cytometry.

Transcript level assessment of angiogenic signatures in CD45+ intratumoral cells with anti-IL-1b and cabozantinib

Top 30 immune genes from Nanonstring I/O 360 gene list for clusters 1, 4, 8, and 12.

Comparison of combination anti-PD1/anti-IL1b and cabozantinib/anti-IL1b treatment.

Assessment of TME changes in MC38 tumors with combination cabozantinib and anti-interleukin-1b immunotherapy.

Evaluation of T cell depletion on the efficacy of combination cabozantinib and anti-interleukin-1b immunotherapy.

Radiation is a known immune modulator that drives both local and systemic immunologic effects. There is increasing interest and investigation into harnessing the pro-immunogenic effects of radiation for patients with metastatic cancer to improve systemic disease control and clinical outcomes. Here, we review fundamental immunology concepts in the context of our current understanding of both the pro-immunogenic and the less well-appreciated immunosuppressive effects of radiation therapy. Our aim is to offer the radiation oncology community a lens into the progress the field has made understanding the complex interaction between tumor-directed irradiation and immune-mediated tumor control, thus promoting further discovery and translation of radio-immuno-oncology innovation.

Bladder cancer represents 3% of all new cancer diagnoses per year. We propose intravesical radionuclide therapy using the β-emitterImage-guided biodistribution of intravesical DBA was investigated in an animal model by radiolabeling DBA with theOur results demonstrated that DBA is contained in the bladder for up to 4 h after intravesical infusion. A single dose of [Our data demonstrates the potential of intravesical [

187 Background: Intraprostatic radiation dose escalation is an area of clinical interest. Dose escalation within the prostate must be balanced with maintaining acceptable dose to the organs at risk, OAR (bladder, rectum, and urethra). Treatment planning therefore requires simultaneous consideration of multiple competing plan optimization goals, for which iterative, interdisciplinary treatment planning tasks may take significant physician, physicist, and dosimetrist time. Semi-automated treatment planning using artificial intelligence has the potential to significantly reduce treatment planning time for technically complex treatments. Methods: A prostate SBRT planning template was created using the Varian ETHOS treatment planning system (TPS) combined with an in-house RapidPlan SBRT prostate model. Prostate dose was prescribed to 36.25 Gy over 5 fractions with 95% coverage to the PTV. To respect standard SBRT normal tissue toxicity constraints while simultaneously escalating intraprostatic dose, the TPS automatically created an intraprostatic boost structure (PTV_SIB), derived from the PTV by excluding OARs with a pre-determined margin. Physicians were trained to perform treatment planning using the prostate SBRT planning template. Treatment planning was performed on 5 unique patients. The time spent from initiation to end of treatment planning and dosimetric parameters were recorded. Results: For each patient, the ETHOS TPS generated two SBRT plans (9 field static IMRT and 3 VMAT arc) with intraprostatic dose escalation in an average of 9.3 minutes [range 8.4-11.8]. Static field and VMAT plans were comparable. PTV_SIB was escalated to above 50 Gy in all cases. Relevant dosimetry for each patient’s static IMRT plan is shown. Conclusions: Physician-driven ETHOS treatment planning was able to produce boosted internal PTV doses using autosegmented volumes. The ETHOS TPS was able to generate dose-escalated plans that reconciled complex OAR and PTV goals within 8-12 minutes. Hence, the ETHOS TPS opens the possibility of rapid physician-driven treatment planning throughput. [Table: see text]

Sarcopenia has been shown to correlate with poor oncologic outcomes, but the optimal method to evaluate sarcopenia and its use as a prognostic tool in glioblastoma multiforme (GBM) remain unclear. We developed a method to reproducibly quantify sarcopenia using the temporalis muscle and correlated sarcopenia with overall survival (OS) in GBM patients. We measured the temporalis muscle width (TMW) on the postoperative radiotherapy CT simulation scan of 87 newly diagnosed GBM patients from 2011 to 2016. All patients successfully completed standard of care radiotherapy to a dose of 6000 cGy in 30 fractions with concurrent temozolomide without treatment breaks. TMW was measured bilaterally on four axial CT slices defined by bony orbit anatomy: orbit ceiling, superior quarter (equidistant between orbit and mid-orbit), mid-orbit, and inferior quarter (equidistant between mid-orbit and orbit floor). For analysis, TMW was dichotomized to either wide or narrow TMW by the median TMW and correlated with OS using Cox regression models. TMW at the orbit inferior quarter significantly correlated with OS. Median OS for wide TMW (> 1.583 cm) was greater than that of narrow TMW: 28.3 mo vs. 22.2 mo (HR (wide/narrow) = 0.42, 95% CI = (0.22, 0.82), p = 0.01), controlling for resection type and MGMT methylation. This effect was pronounced especially in the following patient subsets: females (HR (wide/narrow) = 0.26, 95% CI = (0.07, 0.92), p = 0.04) and patients without MGMT methylation (HR = 0.35, 95% CI = (0.15, 0.81), p = 0.01). Our results suggest sarcopenia and TMW correlate with GBM OS. Prospective validation of sarcopenia is needed to evaluate its potential in determining optimal patient treatment.

TPS5115 Background: Roughly 20%-30% of prostate cancer patients experience biochemical recurrence (BCR), rising prostate-specific antigen (PSA) levels, after definitive therapy with radical prostatectomy (RP) or radiation therapy (RT). The optimal therapy and timing of treatment for BCR is unknown, however, for patients who are not eligible for salvage radiation, androgen deprivation therapy (ADT) is the standard first-line treatment. ADT is an effective therapy but has many acute and long-term toxicities. Novel strategies to reduce ADT exposure and prolong disease control are needed. ADXS-504 is a live attenuated Listeria monocytogenes (Lm)-based immunotherapy consisting of a truncated nonhemolytic fragment of listeriolysin O (tLLO) fused to a total of 24 tumor associated antigens (TAA). ADXS-504 was designed so that nearly 20% of patients with BCR cancer will express at least one of the targeted hotspot mutation peptide antigens and that >90% will express at least one of the TAAs targeted by the sequence-optimized TAA peptide antigens. By combining these shared, commonly expressed antigen targets into a single Lm-based immunotherapy, ADXS-504 provides the potential for a potent, disease-specific approach to treating patients with prostate cancer. In addition to the generation of antigen-specific T cell responses, treatment with Lm-based immunotherapies has been shown to reprogram the tumor microenvironment (TME), by reducing the frequencies and function of immunosuppressive regulatory T cells and myeloid-derived suppressor cells within the TME. A pre-clinical murine model showed that a proto-type Lm-construct could reduce the number of metastases after removal of the primary tumor by eliminating residual tumor cells. We hypothesize that ADXS-504 is safe and promotes anti-tumor immune responses that may delay or prevent the use of ADT in castration sensitive prostate cancer. Methods: This is an open-label dose-escalation phase I trial of ADXS-504 in patients with BCR of prostate cancer previously treated with RP or RT. The study will enroll up to 18 subjects with BCR with a PSA ≥ 0.3 and a PSADT ≥ 4 months without evidence of metastatic disease on traditional CT imaging and bone scans. ADXS-504 will be given by IV every 4 weeks level for 6 study treatments, followed by maintenance therapy given every 12 weeks for 4 doses for overall total of 10 doses of study treatment. DLTs will be evaluated over the first 28 days of treatment. PSA response, PSADT, and adverse events will be summarized. The Kaplan- Meier method will be used to estimate time to PSA progression and rPFS. Immunologic activity will be evaluated by ELISpot analysis, flow cytometry, and multiplex assays and blood samples will be collected for gene sequencing analysis. The study is currently open to enrollment. Clinical trial information: NCT05077098.

Objective: We aim to test the hypothesis that neurovascular bundle (NVB) displacement by rectal hydrogel spacer combined with NVB delineation as an organ at risk (OAR) is a feasible method for NVB-sparing stereotactic body radiotherapy. Methods: Thirty-five men with low- and intermediate-risk prostate cancer who underwent rectal hydrogel spacer placement and pre-, post-spacer prostate MRI studies were treated with prostate SBRT (36.25 Gy in five fractions). A prostate radiologist contoured the NVB on both the pre- and post-spacer T2W MRI sequences that were then registered to the CT simulation scan for NVB-sparing radiation treatment planning. Three SBRT treatment plans were developed for each patient: (1) no NVB sparing, (2) NVB-sparing using pre-spacer MRI, and (3) NVB-sparing using post-spacer MRI. NVB dose constraints include maximum dose 36.25 Gy (100%), V34.4 Gy (95% of dose) Results: Rectal hydrogel spacer placement shifted NVB contours an average of 3.1 ± 3.4 mm away from the prostate, resulting in a 10% decrease in NVB V34.4 Gy in non-NVB-sparing plans (p < 0.01). NVB-sparing treatment planning reduced the NVB V34.4 by 16% without the spacer (p < 0.01) and 25% with spacer (p < 0.001). NVB-sparing did not compromise PTV coverage and OAR endpoints. Conclusions: NVB-sparing SBRT with rectal hydrogel spacer significantly reduces the volume of NVB treated with high-dose radiation. Rectal spacer contributes to this effect through a dosimetrically meaningful displacement of the NVB that may significantly reduce RiED. These results suggest that NVB-sparing SBRT warrants further clinical evaluation. Advances in knowledge: This is a feasibility study showing that the periprostatic NVBs can be spared high doses of radiation during prostate SBRT using a hydrogel spacer and nerve-sparing treatment planning.

BackgroundImmune checkpoint inhibitors (ICIs) are important new therapeutic options for the treatment of malignancy. Existing data on the relative safety of ICI treatment in patients with pre-existing autoimmune disease (AID) are limited.MethodsIn this retrospective study utilizing an oncology medical claims database, we determined the rates of treatment with immunosuppressive agents and hospitalization within 180 days of treatment with ICIs (pembrolizumab, nivolumab, and ipilimumab) in patients both with and without AID. Patients had diagnoses of either malignant melanoma or lung cancer. Immunosuppressive agents evaluated included oral prednisone and intravenous methylprednisolone.Results124 cancer patients with AID and 1896 cancer patients without AID met inclusion criteria for oral prednisone analysis, while 284 patients with AID and 3230 patients without AID met inclusion criteria for all other analyzes. Following treatment with PD-1 inhibitors, rates of treatment with both oral prednisone and intravenous methylprednisolone within 180 days of ICI treatment were significantly increased in the AID group relative to the control group (oral prednisone: 16.7% treatment in AID vs 8.3% in non-AID, p=0.0048; intravenous methylprednisolone: 8.4% treatment in AID vs 3.7% in non-AID, p=0.0012). Rates of hospitalization were significantly increased in melanoma patients with AID relative to melanoma patients without AID following treatment with PD-1 inhibitors (24.1% in AID vs 5.8% in non-AID, pConclusionCancer patients with AID have higher rates of hospitalization and treatment with immunosuppressive agents following treatment with ICI therapy compared with patients with no AID. This suggests that patients with AID may have increased toxicity risk while being treated with checkpoint inhibitor therapy. Further prospective clinical trials are needed to determine safety.

Purpose Radiation therapy (RT) modulates the immune characteristics of the tumor microenvironment (TME). It is not known whether these effects are dependent on the type of RT used. Methods and Materials We evaluated the immunomodulatory effects of carbon-ion therapy (CiRT) compared with biologically equivalent doses of photon therapy (PhRT) on solid tumors. Orthotopic 4T1 mammary tumors in immunocompetent hosts were treated with CiRT or biologically equivalent doses of PhRT. Seventy-two hours after RT, tumors were harvested and the immune characteristics of the TME were quantified by flow cytometry and multiplex cytokine analyses. Results PhRT decreased the abundance of CD4+ and CD8+ T cells in the TME at all doses tested, with compensatory increases in proliferation. By contrast, CiRT did not significantly alter CD8+ T-cell infiltration. High-dose CiRT increased secretion of proinflammatory cytokines by tumor-infiltrating CD8+ T cells, including granzyme B, IL-2, and TNF-α, with no change in IFN-γ. Conversely, high-dose PhRT increased CD8+ T-cell secretion of IFN-γ only. At most of the doses studied, PhRT increased proliferation of immunosuppressive regulatory T cells; this was only seen with high-dose CiRT. Cytokine analyses of bulk dissociated tumors showed that CiRT significantly increased levels of IFN-γ, IL-2, and IL-1β, whereas PhRT increased IL-6 levels alone. Conclusions At low doses, lymphocytes differ in their sensitivity to CiRT compared with PhRT. Unlike PhRT, low-dose CiRT is generally lymphocyte-sparing. At higher doses, CiRT is a more potent inducer of proinflammatory cytokines and merits further study as a modulator of the immunologic characteristics of the TME.

Purpose: Intratumoral immunosuppression mediated by myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) represents a potential mechanism of immune checkpoint inhibitor (ICI) resistance in solid tumors. By promoting TAM and MDSC infiltration, IL1β may drive adaptive and innate immune resistance in renal cell carcinoma (RCC) and in other tumor types. Experimental Design: Using the RENCA model of RCC, we evaluated clinically relevant combinations of anti-IL1β plus either anti-PD-1 or the multitargeted tyrosine kinase inhibitor (TKI), cabozantinib. We performed comprehensive immune profiling of established RENCA tumors via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA sequencing (RNA-seq). Similar analyses were extended to the MC38 tumor model. Results: Analyses via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA-seq showed that anti-IL1β reduces infiltration of polymorphonuclear MDSCs and TAMs. Combination treatment with anti-IL1β plus anti-PD-1 or cabozantinib showed increased antitumor activity that was associated with decreases in immunosuppressive MDSCs and increases in M1-like TAMs. Conclusions: Single-cell RNA-seq analyses show that IL1β blockade and ICI or TKI remodel the myeloid compartment through nonredundant, relatively T-cell–independent mechanisms. IL1β is an upstream mediator of adaptive myeloid resistance and represents a potential target for kidney cancer immunotherapy.

Purpose To implement Velocity-based image fusion and adaptive deformable registration to enable treatment planning for preclinical murine models of fractionated stereotactic radiosurgery (fSRS) using the small animal radiation research platform (SARRP). Methods and Materials C57BL6 mice underwent 3 unique cone beam computed tomography (CBCT) scans: 2 in the prone position and a third supine. A single T1-weighted post-contrast magnetic resonance imaging (MRI) series of a murine metastatic brain tumor model was selected for MRI-to-CBCT registration and gross tumor volume (GTV) identification. Two arms were compared: Arm 1, where we performed 3 individual MRI-to-CBCT fusions using rigid registration, contouring GTVs on each, and Arm 2, where the authors performed MRI-to-CBCT fusion and contoured GTV on the first CBCT followed by Velocity-based adaptive registration. The first CBCT and associated GTV were exported from MuriPlan (Xstrahl Life Sciences) into Velocity (Varian Medical Systems, Inc, Palo Alto, CA). In Arm 1, the second and third CBCTs were exported similarly along with associated GTVs (Arm 1), while in Arm 2, the first (prone) CBCT was fused separately to the second (prone) and third (supine) CBCTs, performing deformable registrations on initial CBCTs and applying resulting matrices to the contoured GTV. Resulting GTVs were compared between Arms 1 and 2. Results Comparing GTV overlays using repeated MRI fusion and GTV delineation (Arm 1) versus those of Velocity-based CBCT and GTV adaptive fusion (Arm 2), mean deviations ± standard deviation in the axial, sagittal, and coronal planes were 0.46 ± 0.16, 0.46 ± 0.22, and 0.37 ± 0.22 mm for prone-to-prone and 0.52 ± 0.27, 0.52 ± 0.36, and 0.68 ± 0.31 mm for prone-to-supine adaptive fusions, respectively. Conclusions Velocity-based adaptive fusion of CBCTs and contoured volumes allows for efficient fSRS planning using a single MRI-to-CBCT fusion. This technique is immediately implementable on current SARRP systems, facilitating advanced preclinical treatment paradigms using existing clinical treatment planning software.

2588 Background: Immune checkpoint inhibitors (ICIs) are potent new cancer therapies but can cause serious immune-related adverse events. Radiation therapy (RT) also induces systemic immunologic effects, and data on the interaction and safety of combining ICIs and RT are limited. Methods: In this retrospective cohort study using a large medical claims database from 2010 to 2017, we ascertained the risk of immunosuppressive steroid therapy as well as the risk of hospitalization within 180 days of treatment with an ICI in patients with diagnoses of malignant melanoma or lung cancer. Patients were stratified by use of RT within 30 days before and after ICI therapy. ICIs included pembrolizumab, nivolumab, and ipilimumab, while immunosuppressive agents included oral prednisone and intravenous methylprednisolone. Results: 2020 patients (218 with RT, 1802 without RT) met inclusion criteria for prednisone analysis, while 3519 patients (361 with RT, 3158 without RT) met inclusion criteria for all other analyses. On univariable analysis, RT was not associated with need for prednisone or methylprednisolone (RR 1.2, 95%CI 0.8-1.9 and RR 1.2, 95%CI 0.7-2.1 respectively). When assessing hospitalization, RT was significantly associated with hospitalization following ICI therapy for certain cancer/drug combinations (RR 1.4, 95%CI 1.2-1.6, p

Introduction/Purpose: Radiation therapy drives pro-inflammatory changes to the tumor microenvironment (TME). We have shown that this may be countered by the induction of immunosuppressive changes that drive therapy resistance. Here, we test the hypothesis that radiation therapy induces immunosuppressive myeloid cells in the TME in an adenosine-dependent fashion. Methods: Using the 4T1 orthotopic breast cancer mouse model, we tested the immunophenotypic changes to the TME by spectral flow cytometry and bulk RNASeq of tumor infiltrating leukocytes (CD45+, TIL) 72-hours after tumor-directed radiation therapy (8 Gy x 1) using the small animal radiation research platform (SARRP, Xstrahl; 220 kV). We cultured human peripheral blood mononuclear cells (PBMCs) with 0.1, 1, and 10uM of adenosine receptor agonist, 5’-N-ethylcarboxamindoadenosine (NECA), in the presence of CD3/CD28 beads. Cytokine quantification of cell-free supernatant was performed 48-hours later using the Meso Scale Discovery platform. Results: Radiation increases the abundance of CD45+CD11b+ myeloid cells (77.4% vs. 84.4%) and decreased CD45+TCRb+ lymphocytes (6.3% vs. 3.6%) in the TME, compared to untreated controls. The reduction of lymphocytes was driven by CD4+ T-cells (5.2% vs 2.7%), including a significant reduction Tregs (1.4% vs 0.63%), with no change in abundance of CD8+ T-cells (0.53% vs 0.43%). The increase in myeloid cells was predominantly influenced by an induction of granulocytic and monocytic myeloid-derived suppressor cells (PMN-MDSCs: 22% vs 34.5%; M-MSCSs: 3.0% vs 10.0%). Radiation caused a decrease in the tumor-associated macrophage population (TAM: 50% vs 31.1%) in the setting of a phenotypic shift to from pro-inflammatory M1-like to immunosuppressive M2-like TAM phenotype: M2-like TAM/M1-like TAM ratio 2.0 vs 13.3. Radiation increased the expression of adenosine 2A receptor (A2AR, log2 fold change 1.13) and A2BR (log2 fold change 0.92) by TILs, but not CD73 that metabolizes AMP to adenosine. The addition of NECA to human PBMCs resulted in significant reduction in effector T-cell cytokines (IL-2, IFN- γ, granzyme B) at all NECA concentrations tested and a dose-dependent decrease in GM-CSF, IL-12p40 and IL-23. There was a significant reduction of IL-1 β, IL-8, IL-12p70 and MCP-1 only at the highest dose (10uM) tested. IL-6 was increased at the intermediate dose (1uM) of NECA with an overall reduction with high-dose NECA. High-dose NECA alone significantly increased both IFN- β and TGF- β. Conclusion: Radiation induces an immunosuppressive TME, increasing the abundance of PMN-MDSCs, M-MDSCs and a shifting the TAM population towards an immunosuppressive phenotype. Radiation increases the expression of adenosine receptors on TILs and treatment of human PBMCs with adenosine receptor agonist resulted in loss of effector T-cell function and suppressive changes to cytokine milieu suggesting a mechanistic link between radiation and adenosine signaling. Citation Format: Shruti Bansal, Mathew Chaimowitz, Casey R. Ager, Aleksandar A. Obradovic, Charles G. Drake, Catherine S. Spina. Adenosine signaling and radiation-induced immunosuppressive changes to the tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-005.

Current in vitro hematopoiesis models fail to demonstrate the cellular diversity and complex functions of living bone marrow; hence, most translational studies relevant to the hematologic system are conducted in live animals. Here we describe a method for fabricating 'bone marrow-on-a-chip' that permits culture of living marrow with a functional hematopoietic niche in vitro by first engineering new bone in vivo, removing it whole and perfusing it with culture medium in a microfluidic device. The engineered bone marrow (eBM) retains hematopoietic stem and progenitor cells in normal in vivo-like proportions for at least 1 week in culture. eBM models organ-level marrow toxicity responses and protective effects of radiation countermeasure drugs, whereas conventional bone marrow culture methods do not. This biomimetic microdevice offers a new approach for analysis of drug responses and toxicities in bone marrow as well as for study of hematopoiesis and hematologic diseases in vitro.

Intratumoral injection of a hydrogel impregnated with radioisotope-labelled catalase and an immunostimulant, along with systemic immune checkpoint blockade, inhibits tumour growth in mouse models of localized cancer and metastatic cancer.

e14140 Background: Immune checkpoint inhibitors (ICIs) are known to cause immune-related adverse events. Patients with autoimmune diseases (AID) were excluded from most ICI clinical trials due to the potentially high risk of adverse effects. Data on the safety of ICIs in patients with a diagnosis of AID is therefore limited. Methods: A retrospective cohort study was conducted using a de-identified large oncology health care and pharmacy claims database with data from March 2010 until April 2017. Patients analyzed had a diagnosis of either melanoma or lung cancer and were treated with either of the anti-PD-1 inhibitors nivolumab or pembrolizumab. We assessed whether patients with AID compared with no AID were more likely to require medical interventions within 180 days of ICI therapy. We determined the percentage of patients receiving oral prednisone, IV methylprednisolone, or were hospitalized, which may represent responses to ICI toxicity. Results: 16.7% (16/96) of patients with either melanoma or lung cancer and AID received oral prednisone treatment within 180 days of ICI treatment, while 8.3% (131/1573) of patients without AID received oral prednisone during the same period. 8.4% (16/190) of patients with AID received IV methylprednisolone compared to 3.6% (79/2190) of patients without AID. Among melanoma patients, 24.1% (13/54) of patients with AID were hospitalized following ICI treatment, compared to 5.8% (28/480) of patients without ICI. Among lung cancer patients, 38.2% (52/136) of patients with AID were hospitalized compared to 31.6% (541/1711) of patients without AID. All comparisons are significant at p < 0.05 except hospitalizations in lung cancer patients. Conclusions: Patients with AID were more likely to receive interventions after ICI treatment that may represent responses to immune-related adverse events, suggesting that patients with AID are at increased risk for toxicity when being treated with ICIs.

The mammalian gut ecosystem has considerable influence on host physiology, but the mechanisms that sustain this complex environment in the face of different stresses remain obscure. Perturbations to the gut ecosystem, such as through antibiotic treatment or diet, are at present interpreted at the level of bacterial phylogeny. Less is known about the contributions of the abundant population of phages to this ecological network. Here we explore the phageome as a potential genetic reservoir for bacterial adaptation by sequencing murine faecal phage populations following antibiotic perturbation. We show that antibiotic treatment leads to the enrichment of phage-encoded genes that confer resistance via disparate mechanisms to the administered drug, as well as genes that confer resistance to antibiotics unrelated to the administered drug, and we demonstrate experimentally that phages from treated mice provide aerobically cultured naive microbiota with increased resistance. Systems-wide analyses uncovered post-treatment phage-encoded processes related to host colonization and growth adaptation, indicating that the phageome becomes broadly enriched for functionally beneficial genes under stress-related conditions. We also show that antibiotic treatment expands the interactions between phage and bacterial species, leading to a more highly connected phage-bacterial network for gene exchange. Our work implicates the phageome in the emergence of multidrug resistance, and indicates that the adaptive capacity of the phageome may represent a community-based mechanism for protecting the gut microflora, preserving its functional robustness during antibiotic stress.

The ever-increasing incidence of antibiotic-resistant infections combined with a weak pipeline of new antibiotics has created a global public health crisis. Accordingly, novel strategies for enhancing our antibiotic arsenal are needed. As antibiotics kill bacteria in part by inducing reactive oxygen species (ROS), we reasoned that targeting microbial ROS production might potentiate antibiotic activity. Here we show that ROS production can be predictably enhanced in Escherichia coli, increasing the bacteria's susceptibility to oxidative attack. We developed an ensemble approach of genome-scale, metabolic models capable of predicting ROS production in E. coli. The metabolic network was systematically perturbed and its flux distribution analyzed to identify targets predicted to increase ROS production. Targets that were predicted in silico were experimentally validated and further shown to confer increased susceptibility to oxidants. Validated targets also increased susceptibility to killing by antibiotics. This work establishes a systems-based method to tune ROS production in bacteria and demonstrates that increased microbial ROS production can potentiate killing by oxidants and antibiotics.

6555Background: While chemotherapy improves outcomes in breast cancer patients, it also increases the risk of neutropenia. There is a need for improved risk prediction models of chemotherapy-induce...

Poor prognosis in neuroblastoma is associated with genetic amplification of MYCN. MYCN is itself a target of let-7, a tumour suppressor family of microRNAs implicated in numerous cancers. LIN28B, an inhibitor of let-7 biogenesis, is overexpressed in neuroblastoma and has been reported to regulate MYCN. Here we show, however, that LIN28B is dispensable in MYCN-amplified neuroblastoma cell lines, despite de-repression of let-7. We further demonstrate that MYCN messenger RNA levels in amplified disease are exceptionally high and sufficient to sponge let-7, which reconciles the dispensability of LIN28B. We found that genetic loss of let-7 is common in neuroblastoma, inversely associated with MYCN amplification, and independently associated with poor outcomes, providing a rationale for chromosomal loss patterns in neuroblastoma. We propose that let-7 disruption by LIN28B, MYCN sponging, or genetic loss is a unifying mechanism of neuroblastoma development with broad implications for cancer pathogenesis.

The searches for drugs that exhibit antineoplastic activity and regulate blood pressure are among the most prevalent and compelling research activities today. Amazingly, there is ample precedence for the antiproliferative action of vitamin-D-related compounds and their role as endocrine suppressors of renin biosynthesis. We have recently synthesized a number of novel calcitriol analogs of the gemini family and originally selected for further studies an epimeric pair related to 19-nor-calcitriol whose 21-methyl group was replaced by a 5,5,5-trifluoro-4-hydroxy-4-(trifluoromethyl)-2-pentynyl group. While maintaining the acceptable calcemic responses, the IC50 concentrations of interferon-gamma release were reduced and the antiproliferative activity and inhibition of renin mRNA expression enhanced. Replacing the geminal methyl groups on the calcitriol-related side chain of these gemini compounds with trideuteriomethyl moieties further boosted the potency in the colon cancer model in mice some 10-fold, reduced NMU-induced breast cancer carcinogenesis in rats and decreased the IC(50) values for renin mRNA inhibition into the pM range.

The active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is an endocrine hormone whose classic role is the maintenance of calcium homeostasis. It is well documented that 1,25(OH)(2)D(3) also has anti-tumor effects on a number of cancers and cancer cell lines including breast, colorectal, gastric, liver, ovarian, prostate, and non-melanoma skin cancers. Included in the anti-tumor activities of 1,25(OH)(2)D(3) are its ability to cause antiproliferation, prodifferentation and decrease angiogenesis. Furthermore, through regulation of the plaminogen activator (PA) system and a class of proteolytic enzymes called matrix metalloproteinases (MMPs), 1,25(OH)(2)D(3) reduces the invasive spread of tumor cells. Because of the calcemic limitations of using 1,25(OH)(2)D(3) as a therapy, we have tested the effects of a novel Gemini vitamin D analogue, Deuterated Gemini (DG), on mouse colorectal cancer. We demonstrated that DG is more potent in reducing tumor volume and mass, compared to control and 1,25(OH)(2)D(3). DG significantly prevented (100% reduction, p0.05) the invasive spread of colorectal tumor cells into the surrounding muscle, and had no effect on serum calcium levels. Thus, DG acts as a selective vitamin D receptor modulator (SVDRM) by enhancing select anti-tumor characteristic 1,25(OH)(2)D(3) activities, without inducing hypercalcemia. Thus, DG shows promise in the development of colorectal cancer therapies.

It has been recognized that people who live at higher latitudes and who are vitamin D deficient are at higher risk of dying from many common cancers including colon cancer. To evaluate the role of vitamin D deficiency on colon tumor growth, Balb/c adult male mice were fed either a vitamin D sufficient or vitamin D deficient diet for 10 weeks. Mice were arranged into groups of six and each animal received subcutaneously 10(4) MC-26 cells in the posterior trunk. The tumor size was recorded daily. By day 9 there was a significant difference in tumor volume between the vitamin D sufficient and vitamin D deficient mice. By day 18 the vitamin D deficient animals had a tumor size that was 56% larger compared to the animals that were vitamin D sufficient. To determine whether treatment with active vitamin D analogs could further decrease colon tumor growth in a vitamin D sufficient state, groups of mice were treated with the novel 19-nor-Gemini compounds. The mice were fed a low calcium diet. Twenty-four hours after tumor implantation, the mice received, three times weekly, one of the vitamin D analogs or the vehicle. The group that received Gemini 1,25-dihydroxy-21(3-hydroxy-3-trifluoromethyl-4-trifluoro-butynyl)-19-nor-20S-cholecalciferol (3) showed a dose-dependent decrease in tumor volume. On day 19, at the dose level of 0.02microg molar equivalents (E), the tumor volume was reduced by 41% when compared to the control group. At the same time point, the hexadeuterated analog 1,25-dihydroxy-21(3-hydroxy-3-trifluoromethyl-4-trifluoro-butynyl)-26,27-hexadeutero-19-nor-20S-cholecalciferol (4), administered at the 10-fold lower dose of 0.002microgE, showed a 52% reduction in tumor volume (p

Wilms Tumor, the most common pediatric kidney cancer, evolves from the failure of terminal differentiation of the embryonic kidney. Here we show that overexpression of the heterochronic regulator Lin28 during kidney development in mice markedly expands nephrogenic progenitors by blocking their final wave of differentiation, ultimately resulting in a pathology highly reminiscent of Wilms tumor. Using lineage-specific promoters to target Lin28 to specific cell types, we observed Wilms tumor only when Lin28 is aberrantly expressed in multiple derivatives of the intermediate mesoderm, implicating the cell of origin as a multipotential renal progenitor. We show that withdrawal of Lin28 expression reverts tumorigenesis and markedly expands the numbers of glomerulus-like structures and that tumor formation is suppressed by enforced expression of Let-7 microRNA. Finally, we demonstrate overexpression of the LIN28B paralog in a significant percentage of human Wilms tumor. Our data thus implicate the Lin28/Let-7 pathway in kidney development and tumorigenesis.

Because of its anabolic effects on muscle, testosterone is being explored as a function-promoting anabolic therapy for functional limitations associated with aging; however, concerns about testosterone's adverse effects on prostate have inspired efforts to develop strategies that selectively increase muscle mass while sparing the prostate. Testosterone's promyogenic effects are mediated through upregulation of follistatin. We show here that the administration of recombinant follistatin (rFst) increased muscle mass in mice, but had no effect on prostate mass. Consistent with the results of rFst administration, follistatin transgenic mice with constitutively elevated follistatin levels displayed greater muscle mass than controls, but had similar prostate weights. To elucidate signaling pathways regulated differentially by testosterone and rFst in prostate and muscle, we performed microarray analysis of mRNAs from prostate and levator ani of castrated male mice treated with vehicle, testosterone, or rFst. Testosterone and rFst shared the regulation of many transcripts in levator ani; however, in prostate, 593 transcripts in several growth-promoting pathways were differentially expressed after testosterone treatment, while rFst showed a negligible effect with only 9 transcripts differentially expressed. Among pathways that were differentially responsive to testosterone in prostate, we identified ornithine decarboxylase (Odc1), an enzyme in polyamine biosynthesis, as a testosterone-responsive gene that is unresponsive to rFst. Accordingly, we administered testosterone with and without α-difluoromethylornithine (DFMO), an Odc1 inhibitor, to castrated mice. DFMO selectively blocked testosterone's effects on prostate, but did not affect testosterone's anabolic effects on muscle. Co-administration of testosterone and Odc1 inhibitor presents a novel therapeutic strategy for prostate-sparing anabolic therapy.

Prolonged antibiotic treatment can lead to detrimental side effects in patients, including ototoxicity, nephrotoxicity, and tendinopathy, yet the mechanisms underlying the effects of antibiotics in mammalian systems remain unclear. It has been suggested that bactericidal antibiotics induce the formation of toxic reactive oxygen species (ROS) in bacteria. We show that clinically relevant doses of bactericidal antibiotics-quinolones, aminoglycosides, and β-lactams-cause mitochondrial dysfunction and ROS overproduction in mammalian cells. We demonstrate that these bactericidal antibiotic-induced effects lead to oxidative damage to DNA, proteins, and membrane lipids. Mice treated with bactericidal antibiotics exhibited elevated oxidative stress markers in the blood, oxidative tissue damage, and up-regulated expression of key genes involved in antioxidant defense mechanisms, which points to the potential physiological relevance of these antibiotic effects. The deleterious effects of bactericidal antibiotics were alleviated in cell culture and in mice by the administration of the antioxidant N-acetyl-l-cysteine or prevented by preferential use of bacteriostatic antibiotics. This work highlights the role of antibiotics in the production of oxidative tissue damage in mammalian cells and presents strategies to mitigate or prevent the resulting damage, with the goal of improving the safety of antibiotic treatment in people.

A declining pipeline of clinically useful antibiotics has made it imperative to develop more effective antimicrobial therapies, particularly against difficult-to-treat Gram-negative pathogens. Silver has been used as an antimicrobial since antiquity, yet its mechanism of action remains unclear. We show that silver disrupts multiple bacterial cellular processes, including disulfide bond formation, metabolism, and iron homeostasis. These changes lead to increased production of reactive oxygen species and increased membrane permeability of Gram-negative bacteria that can potentiate the activity of a broad range of antibiotics against Gram-negative bacteria in different metabolic states, as well as restore antibiotic susceptibility to a resistant bacterial strain. We show both in vitro and in a mouse model of urinary tract infection that the ability of silver to induce oxidative stress can be harnessed to potentiate antibiotic activity. Additionally, we demonstrate in vitro and in two different mouse models of peritonitis that silver sensitizes Gram-negative bacteria to the Gram-positive–specific antibiotic vancomycin, thereby expanding the antibacterial spectrum of this drug. Finally, we used silver and antibiotic combinations in vitro to eradicate bacterial persister cells, and show both in vitro and in a mouse biofilm infection model that silver can enhance antibacterial action against bacteria that produce biofilms. This work shows that silver can be used to enhance the action of existing antibiotics against Gram-negative bacteria, thus strengthening the antibiotic arsenal for fighting bacterial infections.

Vitamin D deficiency has been associated with increased risk of colon cancer in epidemiologic and prospective clinical studies. In vitro and in vivo studies demonstrated that 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and its analogs inhibit colon cancer cell proliferation. Few studies have evaluated the effect of vitamin D deficiency on the development and growth of colon cancer. To assess the antiproliferative effects of 25-hydroxyvitamin D [25(OH)D] and 1,25(OH)2D3 in vitro, we cultured MC-26 (a colon cancer cell line) in the presence of 25(OH)D3 and 1,25(OH)2D3 and performed [3H]thymidine incorporation. The proliferation of MC-26 was significantly inhibited by both 25(OH)D3 and 1,25(OH)2D3. To determine the effect of vitamin D deficiency on colon cancer proliferation, Balb/c mice were rendered vitamin D deficient by feeding them a vitamin D-deficient diet for 3 mo. A group of vitamin D-sufficient mice was given the same diet with supplemental vitamin D. The mice were injected with MC-26 colon cancer cells and the tumors were measured daily for 20 d. Vitamin D-sufficient mice had 40% smaller tumors than vitamin D-deficient mice. The tumors were evaluated for mRNA expression of the vitamin D receptor (VDR) and 25-hydroxvitamin D-1alpha-hydroxylase (1alpha-OHase) by quantitative RT-PCR. The expression of the mRNA for the VDR and the 1alpha-OHase was 37- and 6-fold higher, respectively, in the vitamin D-sufficient mice compared with the vitamin D-deficient mice. We conclude that vitamin D deficiency enhances the growth of colon cancer in mice. The tumor expression of VDR and 1alpha-OHase indicates possible autocrine/paracrine cell growth regulation by vitamin D.

Die antibakterielle Therapie wird immer schwieriger, vor allem gramnegative Erreger zeigen zunehmend Resistenzen auch gegen Reserveantibiotika: Ihre ausere Lipopolysaccharid-Zellmembran schutzt sie vor auseren Einflussen und damit auch vor dem Eindringen von Medikamenten. Eine quasi „antike“ Strategie, die schon Hippokrates beschrieben hat, zieht in diesem Zusammenhang neue Aufmerksamkeit auf sich: Silber kann bei topischer Anwendung die Wundheilung verbessern und Wundinfektionen vermindern. Wie genau das aber funktioniert, war bis jetzt nicht bekannt.

Neuroblastoma is the most frequent extracranial solid tumor in children, accounting for almost 9% of all childhood cancers and close to 15% of childhood cancer deaths. Amplification of MYCN occurs in up to 40% of neuroblastomas, and is a marker of very poor prognosis. Current treatment strategies for MYCN amplified tumors are ineffective and in desperate need of new therapeutic avenues. The LIN28 A and B RNA binding proteins negatively regulate the let-7 family of pro-differentiation/ tumor suppressor miRNAs, enhance the translation of mRNA binding partners, and are emerging as novel onco-proteins. We show here that LIN28B is highly expressed in neuroblastoma, both in human neuroblastoma cell lines and in neuroblastoma patient samples, with an expression pattern similar to MYCN. The LIN28 proteins have been shown to directly regulate MYC, and knockdown of LIN28B in MYCN-amplified human neuroblastoma cells results in markedly reduced MYCN expression, impaired growth, greater differentiation, and reduced tumor size in mouse xenograft studies, indicating an important functional link between LIN28B and MYCN. Through LIN28B/mRNA co-immunoprecipitation we have discovered that LIN28B binds MYCN mRNA, suggesting a novel mechanism of LIN28B contribution to MYCN activity in high-risk neuroblastoma. In addition, we have also discovered that LIN28B is acetylated within its RNA binding domain, and mutational studies have suggested that modulating this post-translational modification may destabilize LIN28B protein stability. Through proteomic analysis of LIN28B -associated proteins, we have identified an acetyl-transferase as a candidate drug target for disruption of the LIN28B/MYCN oncogenic pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2948. doi:1538-7445.AM2012-2948