TSA Cells Research Articles

Abstract 1100: 4-1BBL agonist targeted to fibroblast activation protein α synergizes with radiotherapy in murine breast tumor

Abstract Background. Ionizing radiation (IR) is a double-edge sword for immunotherapy as it may cause both immunosuppressive and immunostimulatory effects. The interactions of IR with the tumor microenvironment (TME) is a key factor for this balance. Fibroblast activation protein (FAP) is expressed on the surface of CAFs in many cancer types and its presence is associated with poor immune response to immune checkpoint blockade in patients. We hypothesize that IR increases FAP expression in CAFs, therefore the combination of IR with targeted immunomodulators such as an agonistic FAP-41BBL bispecific antibody-like fusion protein could enhance the immune-mediated antitumoral effect of these treatments given in combination. Methods: Murine transplantable tumor cells lines (TSA and MC38) were used to investigate increases in FAP expression on CAFs in tumors under irradiation using IHQ and qPCR. Established orthotopic transplanted models were used. We treated bilateral tumor-bearing mice in which only one of the lesions was locally irradiated (2×6Gy) given alone or in combination with a systemic administration of the FAP-41BBL agonistic bispecific construct. Tumor sizes were followed over time and in the cellular composition microenvironment (TME) was assessed by immunochemistry and multiplex tissue immunofluorescence. Selective depletions of immune cell populations were used to delineate the immune system requirements for efficacy.We measured the changes in FAP expression following radiotherapy in engrafted syngeneic TSA and MC38 models using immunohistochemistry (IHC) and qPCR. TSA cells and CAFs were coinjected orthotopically creating bilateral tumor-bearing models, which were irradiated (2x6Gy) only inone of the lesions, either as a single treatment intervention or in combination with systemic administrations of FAP-41BBL. Treatment efficacy was evaluated measuring tumor volume and changes in the TME. Anti -D8 and a neutralizing anti-type-I IFN mAbs were used to define requirements for the efficacy of the radioimmunotherapy combination. Results. Irradiation of TSA-breast cancer tumors showed clear increases of FAP expression levels after local irradiation. The suboptimal radiotherapy regimen chosen failed to control TSA-derived tumors in in which TSA-breast cancer cells had been co-engrafted with CAF. The combination of irradiation and FAP-41BBL resulted in more prominent increases of FAP expression after local irradiation and, importantly, induced durable complete responses in more than 50% of the mice treated with the combination. A role in the therapeutic effect for CD8+ T cells and type-I IFN was uncovered. Robust immune memory was observed in re-challenge experiments. Conclusion. Our data provides a proof-of-concept and mechanistic insights pertaining the therapeutic efficacy of the bispecific FAP-41BBL fusion protein combined with local radiotherapy. Citation Format: Eneko Garate-Soraluze, Irantzu Serrano-Mendioroz, Carlos E de Andrea, Toni Rullan, Christina Claus, Pablo Umana, Christian klein, Ignacio Melero, Maria E. Rodriguez-Ruiz. 4-1BBL agonist targeted to fibroblast activation protein α synergizes with radiotherapy in murine breast tumor [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 1100.

Block of Voltage-Gated Sodium Channels by Aripiprazole in a State-Dependent Manner.

Aripiprazole is an atypical antipsychotic drug, which is prescribed for many psychiatric diseases such as schizophrenia and mania in bipolar disorder. It primarily acts as an agonist of dopaminergic and other G-protein coupled receptors. So far, an interaction with ligand- or voltage-gated ion channels has been classified as weak. Meanwhile, we identified aripiprazole in a preliminary test as a potent blocker of voltage-gated sodium channels. Here, we present a detailed analysis about the interaction of aripiprazole with the dominant voltage-gated sodium channel of heart muscle (hNav1.5). Electrophysiological experiments were performed by means of the patch clamp technique at human heart muscle sodium channels (hNav1.5), heterologously expressed in human TsA cells. Aripiprazole inhibits the hNav1.5 channel in a state- but not use-dependent manner. The affinity for the resting state is weak with an extrapolated Kr of about 55 µM. By contrast, the interaction with the inactivated state is strong. The affinities for the fast and slow inactivated state are in the low micromolar range (0.5-1 µM). Kinetic studies indicate that block development for the inactivated state must be described with a fast (ms) and a slow (s) time constant. Even though the time constants differ by a factor of about 50, the resulting affinity constants were nearly identical (in the range of 0.5 µM). Besides this, aripirazole also interacts with the open state of the channel. Using an inactivation deficit mutant, an affinity of about 1 µM was estimated. In summary, aripiprazole inhibits voltage-gated sodium channels at low micromolar concentrations. This property might add to its possible anticancer and neuroprotective properties.

Abstract 4201: Immune priming with low intensity focused ultrasound and radiation therapy in murine breast cancer

Abstract Introduction: Our long-term goal is to use focal therapy, such as radiation therapy (RT) and ultrasound, to induce stress and engineer the tumor microenvironment (TME) to drive a systemic anti-tumoral immunity, while achieving primary tumor control. Current RT-induced systemic anti-tumoral immunity is dampened by defective tumor antigen presentation resulting in immune escape, an immunosuppressive TME, and the presence of anergic T cells in the immune privileged tumor draining lymph nodes. We have previously developed a proprietary non-ablative low intensity focused ultrasound (LOFU) therapy that induces unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in LOFU-treated cells and reverses T cell anergy in draining lymph nodes in a mouse model of metastatic melanoma. To further extend our findings to other solid tumors, we hypothesized that a combination of acoustic stress and immune priming by non-ablative LOFU, followed by ablative RT will promote the release of heat shock proteins and other damage-associated molecular pattern (DAMP) factors along with tumor-associated antigens for launching a systemic anti-tumor response in a murine breast cancer (TSA). Methods: TSA cells and palpable orthotopic mammary tumors in BALB/c thymic and athymic nude mice were treated with varying parameters of multi-frequency LOFU and RT (8-10 Gy/fraction), over 1-3 consecutive days. Cell surface immunomodulation of TSA cells and tumor infiltrating immune cell populations were analyzed with flow cytometry and tumor volumes were recorded to calculate growth delay along with survival. Results: LOFU-treated TSA cells exhibited an increase in cell surface chaperones, BiP/GRP78, calreticulin, HSP70 and HSP90 at 24 hours post treatment. Furthermore, there was increased secretion of HMGB1, an immunomodulatory DAMP activator for dendritic cells. LOFU+RT-treated tumors had higher numbers of GP70 tetramer-positive CD8+ T cells by day 10 with higher levels of Ki67+ and GzB+ markers. In a dual tumor abscopal model, we observed significantly lower average tumor volumes of both the primary treated, and secondary untreated tumors, 35 days post treatment. There was a trend towards better tumor control and survival in mice with tumors treated with LOFU+RT, when compared to RT alone. Parallel studies in athymic immunodeficient mice failed to regress tumors treated with LOFU+RT. Conclusions: LOFU + RT poses to be a promising combination therapy not only for elimination of primary tumor tissue but also the induction of a tumor-specific immune response. Further optimization of LOFU parameters is underway to maximize the immunogenic effects of LOFU+RT for combination therapy with immunotherapy. Citation Format: Michelle M. Schumacher, Claudia Gutierrez Chavez, Saurabh Singh, Chandra Karunakaran, Sanjay Pandey, Mayank Kumar, Rodney Macedo Gonzales, Chandan Guha. Immune priming with low intensity focused ultrasound and radiation therapy in murine breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4201.

FOXP3 Is an X-Linked Breast Cancer Suppressor Gene and an Important Repressor of the HER-2/ErbB2 Oncogene

(Cell 129, 1275–1286; June 29, 2007) We, the editors of Cell, were contacted by the authors of this article regarding a duplication in Figure 7 that was not identified in the previous 2008 correction. Dr. Yang Liu, the corresponding author, has determined that the error was introduced during revision of the manuscript. In preparing the final figure, three images from the experiment showing that Foxp3 expression inhibits the growth of drug-resistant clones in breast cancer cell lines shown in Figure 7A—SKBR3 cells transfected with Otc cDNA, SKBR3 cells with vector, and MCF-7 cells with Foxp3—were mistakenly inserted into Figure 7D showing that ectopic expression of ErbB2 abrogated Foxp3-mediated repression. These images were incorrectly presented as TSA cells transfected with ErbB2 + pEF1 vector, ErbB2 + Foxp3, and pcDNA6 vector + Foxp3, respectively. The authors no longer have access to the original data, and consequently, a correction is not possible. Given the age of the paper and that the duplication does not compromise the conclusions of the paper, based on the information available to us at this time, we believe that no further action is warranted. FOXP3 Is an X-Linked Breast Cancer Suppressor Gene and an Important Repressor of the HER-2/ErbB2 OncogeneZuo et al.CellJune 29, 2007In BriefThe X-linked Foxp3 is a member of the forkhead/winged helix transcription factor family. Germline mutations cause lethal autoimmune diseases in males. Serendipitously, we observed that female mice heterozygous for the “scurfin” mutation of the Foxp3 gene (Foxp3sf/+) developed cancer at a high rate. The majority of the cancers were mammary carcinomas in which the wild-type Foxp3 allele was inactivated and HER-2/ErbB2 was overexpressed. Foxp3 bound and repressed the HER-2/ErbB2 promoter. Deletion, functionally significant somatic mutations, and downregulation of the FOXP3 gene were commonly found in human breast cancer samples and correlated significantly with HER-2/ErbB2 overexpression, regardless of the status of HER-2 amplification. Full-Text PDF Open Archive

Abstract 2263: Characteristics of the interferon-stimulatory DNA cargo of exosomes produced by irradiated breast cancer cells

Abstract Radiation therapy (RT) used at immunogenic doses (8GyX3) leads to cytosolic accumulation of DNA that binds to the DNA sensor cGAS and activates the cGAMP synthesis, resulting in STING activation and downstream interferon type I (IFN-I) production by cancer cells [1-3]. RT-induced IFN-I is critical for effective anti-tumor immune responses in combination with immune-checkpoint blockade therapy [1]. We have recently demonstrated that tumor-derived exosomes (TEX) secreted by irradiated (8GyX3) TSA murine carcinoma cells (RT-TEX) contain more DNA compared to TEX produced by untreated cells (UT-TEX). Interestingly, only the DNA carried by RT-TEX is capable of STING-dependent activation of recipient dendritic cells (DCs) resulting in CD40, CD80 and CD86 upregulation and IFN-I production. Mice vaccination with RT-TEX, but not UT-TEX, elicits tumor-specific CD8+ T-cell responses that protect mice from tumor development [4]. Here, we tested the hypothesis that not only quantitative but also qualitative differences between the DNA cargo of RT-TEX and UT-TEX may explain its differential ability to activate DCs. To this end, internal DNA purified from TEX and from the cytosolic fraction of the parent TSA cells was characterized for length using Agilent Bioanalyzer. Furthermore, DNA TEX was analyzed by whole-genome sequencing (WGS) and by whole-genome bisulfite sequencing for methylation status. In addition, the percentage of global methylation of DNA of the TSA cells was quantified by 5-methyl cytosine DNA Elisa kit. All experiments were performed in biological triplicates. DNA size analysis revealed an enrichment of DNA fragments with size between 60 and 250 bp in RT-TEX compared to UT-TEX, and in the cytosolic fraction of irradiated compared to untreated TSA cells. The WGS showed that the entire genome was represented in DNA contained within TEX, regardless of the treatment of the parent cells. More than 99% of TEX DNA was of nuclear origin, but mitochondrial DNA was increased in RT-TEX (6.2 fold change, p=0.006). Interestingly, the RT treatment increases the DNA abundance of several regions (median p=0.006). Interestingly, we found reduced levels of methylation in exosomal and total DNA purified from irradiated compared to untreated TSA cells. Our data support the hypothesis that there are potentially important qualitative differences in the DNA cargo of TEX derived from irradiated compared to untreated cancer cells that reflect molecular changes occurring in parent cells. For instance, the enrichment in DNA fragments with size between 60-250 bp in RT-TEX is especially intriguing in light of the recent report that cGAS is optimally activated by this DNA length range [5]. The impact of these qualitative differences in the cargo DNA on activation of the IFN-I pathway in innate immune cells that uptake TEX is under investigation. Identification of DNA signature associated with TEX ability to activate the cGAS/STING pathway could provide a blood-based biomarker for the immunogenic tumor response to radiotherapy. Citation Format: Sheila Spada, Paul Zumbo, Doron Betel, Tuo Zhang, Nils-Petter Rudqvist, Sandra Demaria. Characteristics of the interferon-stimulatory DNA cargo of exosomes produced by irradiated breast cancer cells [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 2263.

Apoptotic caspases inhibit abscopal responses to radiation and identify a new prognostic biomarker for breast cancer patients

ABSTRACTCaspase 3 (CASP3) has a key role in the execution of apoptosis, and many cancer cells are believed to disable CASP3 as a mechanism of resistance to cytotoxic therapeutics. Alongside, CASP3 regulates stress-responsive immunomodulatory pathways, including secretion of type I interferon (IFN). Here, we report that mouse mammary carcinoma TSA cells lacking Casp3 or subjected to chemical caspase inhibition were as sensitive to the cytostatic and cytotoxic effects of radiation therapy (RT) in vitro as their control counterparts, yet secreted increased levels of type I IFN. This effect originated from the accrued accumulation of irradiated cells with cytosolic DNA, likely reflecting the delayed breakdown of cells experiencing mitochondrial permeabilization in the absence of CASP3. Casp3-/- TSA cells growing in immunocompetent syngeneic mice were more sensitive to RT than their CASP3-proficient counterparts, and superior at generating bona fide abscopal responses in the presence of an immune checkpoint blocker. Finally, multiple genetic signatures of apoptotic proficiency were unexpectedly found to have robust negative (rather than positive) prognostic significance in a public cohort of breast cancer patients. However, these latter findings were not consistent with genetic signatures of defective type I IFN signaling, which were rather associated with improved prognosis. Differential gene expression analysis on patient subgroups with divergent prognosis (as stratified by independent signatures of apoptotic proficiency) identified SLC7A2 as a new biomarker with independent prognostic value in breast cancer patients. With the caveats associated with the retrospective investigation of heterogeneous, public databases, our data suggest that apoptotic caspases may influence the survival of breast cancer patients (or at least some subsets thereof) via mechanisms not necessarily related to type I IFN signaling as they identify a novel independent prognostic biomarker that awaits prospective validation.

Abstract 536: NBTXR3 potentiate cancer-cell intrinsic interferon beta response to radiotherapy

Abstract NBTXR3 represent first in class functionalized radioenhancer optimized for efficient tumor binding and sustained local permanence after intratumoral injection. Recent results of phase III in locally advanced Soft Tissue Sarcoma patients established the significant clinical benefits of NBTXR3 activated by radiation therapy compared to radiotherapy alone. Moreover, preclinical studies using CT26 cells have demonstrated that NBTXR3 activated by RT can induce an anti-tumor immune-response and the abscopal effect. Radiotherapy (RT) can induce T cell-mediated anti-tumor immune responses by multiple mechanisms which include enhancing the recruitment of Batf3-dependent cross-priming dendritic cells (DCs). The latter is contingent on tumor-intrinsic IFN-I induction by cytosolic dsDNA via cGAS/STING pathway, a process that is regulated by the upregulation of Trex1 in a RT dose-dependent fashion (Nat Commun 2017; 8:15618). Here, we utilized the murine TSA breast cancer cell line to test the hypothesis that NBTXR3 strengthens the pro-immunogenic effect of local RT by potentiating tumor cells IFN-I response. For in vitro experiments, TSA cells were exposed to NBTXR3 (800uM) 16 hours prior to RT delivered by a 220-kV source (SARRP) at a dose rate of 283.2 cGy/min. Secreted IFNb in the supernatants was measured by ELISA. Pre-treatment with NBTXR3 enhanced TSA cell radiosensitivity as determined in clonogenic assays. Cell death, as measured by loss of plasma membrane integrity was accelerated by NBTX3 after irradiation of TSA cells with 6 and 8Gy after 24 hours of treatment. NBTXR3 enhanced the secretion of IFNb by irradiated cells. Data suggest that NBTXR3 may enhance the effectiveness of radiation and improve tumor immunogenicity, likely via the induction of IFN-I. The role of the cGAS/STING pathway in the effects of NBTX3 are under investigation in vitro and in vivo. Citation Format: Maria E Rodriguez-Ruiz, Karsten Pilones, Camille Daviaud, Jeffrey Kraynak, Audrey Darmon, Sebastien Paris, Sandra Demaria. NBTXR3 potentiate cancer-cell intrinsic interferon beta response to radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 536.

Abstract B199: Potent induction of immunogenic cell death by PT-112

Abstract Purpose: To characterize the ability of PT-112 to induce immunogenic cell death (ICD) in vitro and in vivo. Background: PT-112 is a novel chemical entity consisting of a platinum core conjugated to a pyrophosphate and diaminocyclohexane groups. In vitro studies demonstrated that PT-112 has both cytostatic and cytotoxic effects on human cancer cells, two effects that are seen in the absence of robust binding to nuclear DNA. Accordingly, PT-112 potency is largely unaffected by functional DNA repair pathways, suggesting that PT-112 operates with mechanisms that differ from conventional DNA-damaging chemotherapies. PT-112 is currently under phase I/II clinical development in patients with solid tumors and hematologic malignancies, both as monotherapy and in combination with a PD-L1 inhibitor. Interim reports have established encouraging tolerability and signals of single-agent anticancer activity. Previous work with human colorectal cancer HCT-116 cells demonstrated that PT-112 causes the release of ICD-relevant damage-associated molecular patterns (DAMPs). ICD is a particularly relevant form of cell death for cancer therapy as it can drive tumor-targeting immune responses of clinical relevance. Here, we tested the ability of PT-112 to induce bona fide ICD in murine breast carcinoma TSA cells by in vitro DAMP release studies and in vivo gold-standard vaccination experiments. Methods: TSA cells were exposed to PT-112, cisplatin (negative ICD control) or mitoxantrone (MTX, positive ICD control) at different concentrations for 24 and 48 hours, followed by cytofluorometric assessment of cell number, viability, cell cycle distribution and exposure of the membrane-assocaited DAMP calreticulin (CRT). In addition, culture supernatants were assayed for the release of two other DAMPs, namely ATP and HMGB1, by luminometric tests and ELISA, respectively. BALB/c mice were vaccinated with TSA cells succumbing to optimal PT-112 (or MTX and cisplatin, as positive and negative controls, respectively) concentrations or mock vaccinated (with PBS). After 14 days this was followed by a rechallenge with living TSA cells and scoring of tumor-free survival (TFS) and tumor growth. Results: In vitro, PT-112 caused a robust cytostatic and cytotoxic effect on TSA cells that was accompanied by robust CRT exposure, ATP secretion and HMGB1 release. In line with such an ICD-compatible profile, 10/10 mice vaccinated with PT-112-treated TSA cells were tumor-free after the 1st rechallenge, and also rejected a subsequent challenge performed 1 month later, suggesting the establishment of long-term immunologic anticancer memory. As expected, cisplatin-treated TSA cells failed to mediate a similar effect (TFS at D7 after rechallenge: 10%), as did the mock vaccination arm (TFS at D7: 20%). MTX-treated cells conferred partial protection (TFS at D7: 40%), and the developing tumors had significantly delayed kinetics as compared to tumors evolving in mock-vaccinated mice. Conclusions: In the TSA model system of ICD vaccination, PT-112 is superior to MTX at inducing ICD and the associated establishment of immunologic anticancer memory in vivo. These data provide a robust rationale to combining PT-112 with checkpoint inhibitors in the clinic, to achieve maximal therapeutic effects potentially coupled with long-term protection from metastatic reactivation. Citation Format: Takahiro Yamazaki, Tyler David Ames, Lorenzo Galluzzi. Potent induction of immunogenic cell death by PT-112 [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B199.

Abstract A150: Enhancing abscopal responses to radiation therapy by manipulating autophagy

Abstract Background: Macroautophagy (autophagy) is an evolutionary conserved cellular mechanism culminating with the lysosomal degradation of dispensable, damaged or potentially toxic cytoplasmic structures (e.g., permeabilized mitochondria). Autophagy helps cancer cells to adapt to harsh environmental conditions and to resist therapy. However, autophagy is also key for multiple steps of the anticancer immune response. Thus, whether autophagy should be inhibited or activated in the context of cancer therapy remains debated (Rybstein et al., Nat Cell Biol 2018). Since autophagy has been shown to play a key role in removal of cytosolic DNA, which is one mechanism leading to type I interferon (IFN) secretion, and since type I IFN is required for systemic immune responses activated by radiation therapy (RT), we asked the question as to whether selectively inhibiting autophagy in cancer cells may boost the ability of RT to initiate anticancer immunity. Methods/Tools: CRISPR/Cas9 technology was used to render mouse mammary carcinoma TSA and EO771 cells autophagy-deficient and chemical inhibitors of autophagy were also employed. Autophagy-competent versus –deficient systems were characterized for autophagy proficiency (by immunoblotting), growth (in vitro and in vivo), resistance to cell death induced by starvation, chemotherapy and RT (by multicolor flow cytometry and clonogenic assays) and production of type I IFN (by PCR and ELISA). Abscopal responses have been assessed in vivo. Results: In line with previous observations, autophagy inhibition reduced the growth of mouse mammary carcinoma cells, in vitro and in vivo, limited their clonogenic potential (at baseline) and increased their sensitivity to multiple stressors. Moreover, pharmacologic and genetic autophagy inhibition increased the capacity of mouse mammary carcinoma cells to secrete type I IFN in response to radiation. Finally, immunocompetent mice bearing syngeneic autophagy-deficient mouse mammary carcinoma cells mounted improved abscopal responses to RT (in the context of CTLA4 blockade) as compared to immunocompetent mice bearing syngeneic autophagy-competent cells, as determined by growth inhibition of a distant, non-irradiated, autophagy-competent lesion. Perspectives: We will test the innovative hypothesis that selective autophagy inhibition in cancer cells may synergize with autophagy activation at the whole-body level (by nutrient restriction or physical exercise), hence enabling superior therapeutic responses to radiation. Citation Format: Marissa Rybstein, Takahiro Yamazaki, Aitziber Buque Martinez, Lorenzo Galluzzi. Enhancing abscopal responses to radiation therapy by manipulating autophagy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A150.

PTEN Signaling at the Tumor: Immune Interface

The phosphatase and tensin homolog, PTEN, modulates a wide array of signaling processes that control cell metabolism and proliferation. Preclinical studies in gliomas and prostate carcinomas found that loss of the Pten gene imparts a senescent phenotype with diminished response to radiation and suppressed anti-tumor immunity. We utilized two tumor models of Pten mutation and analyzed immunologically relevant phenotypic changes, including after administration of radiation. Cell cycle distribution was analyzed on HeLa cells with PTEN knockdown following a single fraction of 10 Gy radiation. To further assess radiation responsiveness, Pten wild type and knockout mouse TSA breast cancer cells were treated with 5 Gy of radiation and evaluated for colony formation. HMGB1 release was measured as a surrogate of immunogenic cell death: specifically, TSA cells expressing a recombinant HMGB1-RFA fusion protein were transfected with Pten siRNA and treated with a single fraction of radiation, after which secreted levels of the fusion protein were quantified. Additionally, Pten+/+ and Pten-/- TSA cells were treated with a single fraction of radiation, and PD-L1 expression was analyzed by qPCR 24 hours later. Cell cycle analysis revealed a defect of the DNA damage checkpoint in PTEN knockdown HeLa cells. Following radiation, 64% of control HeLa cells were arrested at G2/M compared to only 34% of PTEN knockdown cells. Furthermore, irradiated Pten-/- TSA cells showed more resilient colony formation than Pten+/+ cells. The analysis of HMGB1 as a surrogate of immunogenic cell death demonstrated a 27% relative decrease of secreted HMGB1 following a single fraction of high dose radiation in Pten-/- cells, as compared to the Pten+/+ population (P < 0.0001). In addition, gene expression analysis of Pten+/+ and Pten-/- TSA cells revealed a 2-fold baseline increase in PD-L1 expression in Pten knockout cells. PTEN loss is a frequent alteration in cancer. We found that PTEN deficiency in tumor cells results in a reduced checkpoint response and more resilient colony formation following radiation, suggesting increased resistance. Furthermore, decreased secretion of HMGB1 and increased gene expression of PD-L1 in the Pten-/- cells are predictive of an immune resistant phenotype. Given these features, PTEN deficient tumor cells may be more prone to survive radiation therapy, even with an accumulating DNA damage. This trait, together with their expression of PD-L1, may suggest utility in employing anti PD-L1 immunotherapy. Further characterization of the impact of PTEN mutation on tumor responses to radiation and immunotherapy will help guide future treatment strategies in patients with associated mutations.

High Linear Energy Transfer Particle Irradiation Leads to Increased Expression of In Vitro Markers of Immunogenic Cell Death

Radiotherapy (RT) fosters a form of tumor cell death that results in dendritic cell activation, effector T-cell cross priming/tumor trafficking, and the formation of a tumor cell/T-cell immune response. This type of immunogenicity is comprised of the release of immune modulating signals, which include the release of high mobility group box 1 (HMGB1), ATP, and the translocation of calreticulin to the cell surface. Previous studies have demonstrated a positive correlation between markers of immunogenic cell death (ICD) and radiation dose with X-rays. Less is known about the effect of higher energies using charged particle irradiation and ICD. In this study, we explore whether varying linear energy transfer (LET) greater than 60 keV/μm with helium ion irradiation would impact immunogenicity in tumor cells as measured by levels of calreticulin translocation, ATP release, and HMGB1 release. Reporter cell lines designed to detect ATP luminescence (RLUs), red fluorescence protein tagged HMGB1 (relative fluorescent units [RFUs]), and cell surface calreticulin (percentage of calreticulin positive cells [%CRT+]) were derived from TSA (murine mammary carcinoma; syngeneic to BALB/c mice) cells. Prior to irradiation, the cells were seeded on six micron thick Mylar attached to 1.5 cm metal rings. The cells were then irradiated at the Radiological Research Accelerator Facility (RARAF) through a Track Segment Charged-Particle Irradiator. ATP and HMGB1 levels were measured 72 hours after RT and calreticulin was measured 24 hours after RT. Various doses of 250kV X-rays and LETs of helium ions were delivered and levels of these tumor ICD markers were determined. For ATP measurements, when compared to non-irradiated controls, cells irradiated with 5 and 20 Gy X-rays and with 5 Gy 65, 80, 110, and 160 keV/μm helium ions resulted in RLU fold-changes of 1.00 ± 0.05 (non-irradiated), 2.33 ± 0.08, 3.27 ± 0.19, 3.24 ± 0.10, 3.99 ± 0.33, 4.12 ± 0.32, and 2.91 ± 0.16 respectively. For HMGB1 measurements, when compared to controls, cells irradiated with 5 and 20 Gy X-rays and with 5 Gy 65, 80, 110, and 160 keV/μm helium ions resulted in RFU fold-changes of 1.00 ± 0.02 (non-irradiated), 1.43 ± 0.03, 1.72 ± 0.03, 1.97 ± 0.06, 1.64 ± 0.03, 1.61 ± 0.04, and 1.51 ± 0.03, respectively. For calreticulin measurements, when compared to controls, cells irradiated with 5 and 20 Gy X-rays and with 5 Gy 65, 80, 110, and 160 keV/μm helium ions resulted in %CRT+ fold-changes of 1.00 ± 0.12 (non-irradiated), 2.32 ± 0.15, 3.07 ± 0.32, 3.16 ± 0.17, 3.01 ± 0.12, 2.74 ± 0.39, and 1.61 ± 0.15, respectively. Immunogenicity of tumor cells as measured by immune cell death signals increases with high LET ion irradiation when compared to low LET X-ray irradiation at the same dose. These marker levels appear to decrease at LETs greater than 110 keV/μm. Further investigation is warranted on the effects of high LET charged particle irradiation on the tumor immune response.

State-dependent block of voltage-gated sodium channels by the casein-kinase 1 inhibitor IC261.

Background and Purpose IC261 (3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one) has previously been introduced as an isoform specific inhibitor of casein kinase 1 (CK1) causing cell cycle arrest or cell death of established tumor cell lines. However, it is reasonable to assume that not all antitumor activities of IC261 are mediated by the inhibition of CK1. Meanwhile there is growing evidence that functional voltage-gated sodium channels are also implicated in the progression of tumors as their blockage suppresses tumor migration and invasion of different tumor cell lines. Thus, we asked whether IC261 functionally inhibits voltage-gated sodium channels. Experimental Approach Electrophysiological experiments were performed using the patch-clamp technique at human heart muscle sodium channels heterologously expressed in human TsA cells. Key Results IC261 inhibits sodium channels in a state-dependent manner. IC261 does not interact with the open channel and has only a low affinity for the resting state of the hNav1.5 (human voltage-gated sodium channel; Kr: 120μM). The efficacy of IC261 strongly increases with membrane depolarisation, indicating that the inactivated state is an important target. The results of different experimental approaches finally revealed an affinity of IC261 to the inactivated state between 1 and 2μM. Conclusion and Implications IC261 inhibits sodium channels at a similar concentration necessary to reduce CK1δ/ε activity by 50% (IC50 value 1μM). Thus, inhibition of sodium channels might contribute to the antitumor activity of IC261.

Abstract 4033: Adenosine regulates radiation therapy-induced antitumor immunity

Abstract Radiation-induced immunogenic cell death (ICD) is a key mechanism whereby local radiation therapy (RT) can elicit anti-tumor immune responses and synergize with immune checkpoint inhibitors in enhancing tumor responses. ATP, which is an essential signal of ICD, activates tumor-resident dendritic cells (DCs) promoting their ability to cross-present tumor-derived antigens to T cells. Interestingly, while release of ATP by RT is dose-dependent (Golden et al., OncoImmunology 2014), a large RT dose of 20 Gy was not effective in inducing anti-tumor T cells and synergize with anti-CTLA-4 (Dewan et al., Clin Cancer Res 2009). Extracellular ATP is rapidly catabolized to adenosine (ADO) by ectonucleotidases CD39 and CD73, which are expressed on tumor cells and immune cells. ADO has immunosuppressive effects, inhibiting DC- and effector T cell-activation, while promoting regulatory T cells (Tregs). Here, we tested the hypothesis that conversion of ATP to ADO hinders generation of effective anti-tumor immunity by high dose RT. Mice were inoculated s.c. with TSA breast cancer cells or MCA38 colon cancer cells on day 0 and assigned to treatment with: (1) control mAb; (2) anti-CD73 (TY/23); (3) RT (4) RT+TY/23. TY/23 (200 μg) was administered i.p. on day 11, 14, 17 and 20. RT was given locally as single 20 Gy dose on day 12. On day 18, some tumors were harvested for flow cytometry analysis of DC and T cells. Mice were monitored for tumor progression. HPLC was used to measure ADO levels in supernatants from 24 h co-cultures of bone marrow-derived DCs and irradiated TSA cells.In vitro, antibody blockade of CD73, the rate-limiting enzyme in the generation of ADO, reduced the levels of ADO in the supernatant and restored the activation of DCs cultured with irradiated TSA cells. In irradiated tumors, anti-CD73 reduced the percentage of Tregs within the tumor-infiltrating CD4+ T cell population (7.9±2.5% in RT+TY/23 vs 20±0.8% in RT, p&amp;lt;0.01) while increasing CD8+ T cells (38.3±0.1% in RT+TY/23 vs 17.3±4% in RT, p&amp;lt;0.05). Among intratumoral DCs, the CD8a+ DC subpopulation was increased after CD73-blockade (37.9±15.7% in TY/23+RT vs 11.3±4.9% in RT, p&amp;lt;0.01). Importantly, in irradiated tumors, anti-CD73 enhanced expression of activation markers CD40 on CD8a+ DCs (MFI: 218±1 in RT+TY/23 vs 54±41 in RT, p&amp;lt;0.05) and CD69 on CD8+ T cells (MFI: 513±126 in RT+TY/23 vs 148±59 in RT, p&amp;lt;0.01). Furthermore, tumor-bearing mice treated with RT in combination with anti-CD73 had a significantly delayed tumor progression (p&amp;lt;0.05) and prolonged survival (p&amp;lt;0.01) compared to mice receiving RT alone. Anti-CD73 given alone had no effect on tumor growth. Our data show that adenosinergic signaling regulates the ability of RT to induce anti-tumor immunity, affecting activation of both DCs and effector T cells. ADO blockade may represent a promising strategy to enhance the immunogenicity of irradiated tumors by improving the ability of RT to induce in situ tumor vaccination. Citation Format: Erik Wennerberg, Aranzazu Mediero, Tuere Wilder, Silvia Formenti, Bruce Cronstein, Sandra Demaria. Adenosine regulates radiation therapy-induced antitumor immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4033.

Abstract A21: Adenosine is a rheostat for radiation therapy-induced immune activation

Abstract Background: Administration of anti-CTLA-4 antibodies has shown clinical benefit in a small percentage of cancer patients. However, most patients are unresponsive to anti-CTLA-4 antibody treatment, due to immunosuppressive mechanisms and/or the absence of sufficient spontaneous anti-tumor T cells that can be activated by CTLA-4 blockade. We have previously shown that radiotherapy (RT) induces immunogenic tumor cell death and priming of anti-tumor T cells making tumors susceptible to anti-CTLA-4 treatment. RT also triggers the release of ATP in the tumor microenvironment, which has proinflammatory effects. However, ATP is rapidly converted into adenosine, a pleiotropic immunosuppressive mediator, by ectonucleotidases CD39 and CD73 expressed on cancer cells and tumor stromal cells. We hypothesized that adenosine generation in the tumor microenvironment acts as a rheostat modulating RT ability to prime effective anti-tumor immunity. To test this hypothesis we used a mouse tumor model where we have previously characterized two RT regimens that were efficient (8Gy x 3) or inefficient (20 Gy) in inducing anti-tumor immunity in combination with anti-CTLA-4 antibodies. Method: BALB/c mice were inoculated s.c. with 1 x 105 TSA cells, a poorly immunogenic breast cancer cell line on day 0. Anti-CD73 mAb TY/23 (200 µg) was administered i.p. every 4 days starting on day 11. RT was given locally to the tumor as a single 20 Gy dose on day 12 or in three doses of 8 Gy each given on day 12, 13 and 14. At day 19, tumors were harvested and infiltrating immune cells were isolated for analysis of DC and T cell phenotype by flow cytometry. In another experiment, mice were randomly assigned (n=5/group) to be treated with: (1) Isotype control mAb (2) TY/23 (3) TY/23 + anti-CTLA-4 mAb 9H10 (4) RT 20 Gy (5) RT 20 Gy + TY/23 (6) RT 20 Gy + 9H10 (6) RT 20 Gy + TY/23+ 9H10. RT was given on day 12 and mAbs were administered i.p. on day 11, 14, 17 and 20 after tumor inoculation. Mice were monitored for tumor growth. Results: In tumors that had received 8GyX3 RT there was a four-fold increase in CD70 expression (MFI 436 ± 72 in 8GyX3 vs. 110 ± 30 in control, p&amp;lt;0.05) on CD8α+ dendritic cells (DCs), which are responsible for tumor-antigen cross-presentation. In addition, CD8+ T cells showed a two-fold increase in PD-1 expression (MFI 550 ± 22 in 8GyX3 vs. 271 ± 35 in control, p&amp;lt;0.01) and 1.6-fold increase in CD69 expression (MFI 351 ± 95 in 8GyX3 vs. 225 ± 40 in control), indicating increased activation of both DC and T cells in tumors treated with 8GyX3 RT. In contrast, there was no evidence of activation of DC and T cells infiltrating tumors treated with 20Gy RT. However, when 20Gy RT was given in combination with TY/23 to block adenosine generation, DC and T cells showed an increase in expression of activation markers comparable to what was observed in tumors treated with 8GyX3. Interestingly, the frequency of intratumoral CD4+CD25+FoxP3+ regulatory T cells (Treg) was increased in mice receiving TY/23 + 8GyX3 (62% increase, p&amp;lt;0.05) compared to TY/23 alone, suggesting that the inflammation induced by RT may trigger Treg recruitment to the tumor when it is not controlled by adenosine. TY/23 and 9H10 given alone or in combination did not have any effect on tumor growth. However, they improved tumor inhibition obtained with 20 Gy RT (Two-way ANOVA: p=0.08 for RT+anti-CD73 and p&amp;lt;0.05 for RT+9H10 vs. RT alone). Moreover, blockade of both CD73 and CTLA-4 in combination with 20Gy RT further improved tumor control resulting in complete tumor regression in 2/5 mice (p&amp;lt;0.01 for RT+TY/23+9H10 vs. RT alone). Conclusions: Data indicate that the ability of RT to promote immune activation is dependent on the radiation regimen employed and is regulated by adenosine generation by the ectonucleotidase CD73. Importantly, data suggest that blocking CD73 can improve the ability of RT to synergize with immunotherapy. Citation Format: Erik Wennerberg, Noriko Kawashima, Sandra Demaria. Adenosine is a rheostat for radiation therapy-induced immune activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A21.

Open channel block of NMDA receptors by diphenhydramine

Background and purposeDiphenhydramine is a well known H1-receptor antagonist that plays a major role in clinical practice. Nowadays, diphenhydramine is primarily applied to prevent nausea but also its sedative and analgesic effects are of clinical importance. As other drugs mediating sedative and analgesic properties partly operate via the inhibition of glutamate receptors, we tested the hypothesis that diphenhydramine, as well interacts with excitatory ionotropic glutamate receptors. Experimental approachElectrophysiological patch-clamp experiments were performed on glutamate receptors which were heterologously expressed in human TsA cells. Key resultsDiphenhydramine inhibits NMDA-mediated membrane currents in a reversible and concentration-dependent manner at clinically relevant concentrations. The inhibition occurred in a noncompetitive manner. Diphenhydramine did not compete with NMDA or glycine for their binding sites and half-maximal inhibition was obtained around 25 μM diphenhydramine, independent of the subunit composition. The inhibition was caused by a classical open channel blocking mechanism and varied strongly with the membrane potential. Our results suggest that diphenhydramine most probably interacts with the Mg2+ binding site or a very closely related area of the channel pore. Conclusion and implicationsThe data presented here provide evidence that the NMDA receptor antagonism of diphenhydramine contribute to its sedative and potentially LTP-related effects like analgesia and amnesia.

Abstract 2493: Fractionated but not single dose radiation is an optimal adjuvant for in situ tumor vaccination

Abstract Local radiotherapy (RT) has the potential to generate an in situ tumor vaccine by inducing an immunogenic cell death (ICD) of the cancer cells. In vitro, generation of the signals that define ICD is radiation dose-dependent. However, in vivo we have previously shown that radiation doses of 8 and 6 Gy given in 3 to 5 consecutive days were more effective at inducing anti-tumor T cell responses in combination with anti-CTLA-4 antibody than a single larger dose of 20 Gy (Dewan et al., Clin Cancer Res 2009). To understand the mechanisms underlying the different outcome obtained with fractionated versus single dose RT, TSA tumors growing in syngeneic immunocompetent BALB/c mice were harvested at 4, 24 and 48 hrs post irradiation with 8 Gy X 3 or 20 Gy X 1 for analysis of gene expression using Agilent mouse mRNA Microarray Kit (V2). Subsequent data were analyzed using Ingenuity Pathway Analysis (IPA) and gene ontology classification. Expression of key immune genes in TSA cells irradiated in vitro was assessed by RT-qPCR. Tumor-infiltrating dendritic cells (TIDC) were analyzed 5 days after the last radiation exposure by flow cytometry. IFNβ production by irradiated TSA cells was measured by Procarta™ immunoassay. Over 100 immune response genes were differentially expressed in tumors irradiated with 8Gyx3 but not 20Gyx1, with a dominant type I interferon (IFN) response at 4 and 24 hours, which was confirmed by RT-qPCR. Tumor infiltration by CD8α+ dendritic cells (DC), which are the subset of DC cross-presenting tumor cell-derived antigens, was only slightly increased by 20Gyx1 but markedly increased by 8Gyx3. Moreover, DCs present in tumors treated with 8Gyx3 showed a significant upregulation of activation markers CD40 and CD70. Importantly, the in vitro setting (devoid of an immune infiltrate) demonstrated expression of IFNβ and downstream immune genes, including chemokines CXCL9, CXCL10 and CXCL11 by TSA cells irradiated with 8Gyx3 but not 20Gyx1. Production of IFNβ by TSA cells was confirmed with an increased secretion of IFNβ in the supernatant only after 8Gyx3 irradiation. Overall, data indicate that fractionated RT can mimic, at least in part, a viral infection and activate canonical defense pathways in neoplastic epithelial cells with induction of type-I IFN. In vivo this leads to recruitment and activation of DC cross-presenting tumor antigens. Thus, fractionated-RT acts as an optimal immune adjuvant for in situ tumor vaccination. We propose that repeated cycles of RT-induced cell death may better mimick cycles of viral infection and are currently defining the molecular mechanisms involved. Results are important for understanding the determinant of response in patients currently treated wit RT and anti-CTLA-4 in clinical trials at our institution. Citation Format: Claire Isabelle Vanpouille-Box, Molykutty Aryankalayil, Karsten A. Pilones, Silvia C. Formenti, Norman Coleman, Sandra Demaria. Fractionated but not single dose radiation is an optimal adjuvant for in situ tumor vaccination. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2493. doi:10.1158/1538-7445.AM2015-2493

Abstract 3329: Ionizing radiation modifies the molecular composition of exosomes derived from breast cancer cells

Abstract Metastatic breast cancer fosters an immunosuppressive tumor microenvironment (TME). Local ionizing radiotherapy (RT) has the potential to convert the tumor into an in situ vaccine by facilitating efficient uptake of tumor neoantigens by dendritic cells and enhancing antigen presentation to promote robust effector T cell responses. Exosomes are microvesicles (30-100nm) released from living cells that shuttle and transfer selected intracellular components, including cytokines, growth factors, mRNA, and miRNA. Tumor-derived exosomes (TEX) allow for a sophisticated means of communication with a variety of cells, including immune cells, within the TME. We hypothesized that TEX released from irradiated tumors may play a role in altering the susceptibility of tumor cells to immune-mediated rejection. To determine if RT changes TEX composition, we treated mouse mammary carcinoma cells TSA in vitro with sham RT, 1 dose of 20Gy, or 3 fractions of 8Gy given in three consecutive days (8Gyx3). Cells were transferred to exosome-depleted media following RT and cell-free supernatant was collected 48hr later. TEX were isolated using differential ultracentrifugation and purified with a sucrose gradient. Electron microscopy was used to confirm the expected size and morphology. TEX were lysed for identification of proteins using label-free quantitation mass spectrometry (LFQ-MS) followed by MS/MS analyses. To characterize miRNA signatures of TEX and their parent cells, RNA was isolated for nanoString miRNA expression assay, which includes a panel of 578 mouse miRNAs. Normalized results were analyzed with MultiExperiment Viewer. LFQ-MS revealed that TEX from 20Gy- and 8Gyx3-irradiated TSA shared 10 unique proteins not present in TEX from non-irradiated TSA. In addition, 17 unique proteins were present only in TEX from 8Gyx3-treated TSA. Among them were proteins involved in T cell development, MHC class I peptide processing, and proinflammatory lipid signaling. Radiation induced downregulation of 73% of miRNAs expressed in untreated TSA cells. Many of the changes in miRNA expression seen in irradiated cells were also seen in the TEX derived from these cells. Interestingly, unique expression patterns emerged with different miRNA subsets expressed in TEX depending on RT regimen used. Interesting candidates are being analyzed for functional relevance. This data suggests that irradiation of tumor cells results in changes in protein and miRNA composition of TEX, with differences dependent on the RT regimen used. We have previously shown that 8Gyx3 RT synergized with anti-CTLA-4 antibody and induced an abscopal effect in mice, while 20Gy did not (Dewan et al, Clin Cancer Res, 2009). We are testing whether TEX contain biomarkers that define the ability of RT regimens to synergize with immunotherapy. Exosome-mediated communication networks may provide a new therapeutic target for treatment of metastatic cancer. Citation Format: Julie M. Diamond, Jessica R. Chapman, Beatrix M. Ueberheide, Sandra Demaria. Ionizing radiation modifies the molecular composition of exosomes derived from breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3329. doi:10.1158/1538-7445.AM2015-3329

Lactoferrin-loaded contact lenses: eye protection against oxidative stress.

Tear fluid contains antioxidative compounds, vitamin C, glutathione, superoxide dismutase, and lactoferrin (LF), which protect the corneal epithelium from the effects of ultraviolet irradiation, direct airflow, and chemical agents. However, these natural defenses against oxidative stress can decrease, favoring the development of anterior eye disorders, such as keratoconus, dry eye, and Sjögren syndrome. LF is an iron-binding glycoprotein, present in mammalian secretions such as tears and milk, endowed with different physiological functions such as antimicrobial, antiviral, and antioxidant activities. In this work, we studied the capability of different soft contact lenses to adsorb and release LF to restore cellular viability in oxidative stress conditions. Three types of contact lenses (filcon V, galyfilcon A, and filcon IB) were loaded with LF and then incubated with TsA or human corneal epithelial primary cells. After oxidative stress induction with 250 μM or 125 μM H2O2, cell viability was evaluated. Data showed that the highest quantity of LF loaded in contact lenses was between 61 μg (for filcon V) and 39 μg (for filcon IB); the release was between 49% and 100% of protein adsorbed. LF released from contact lenses maintained its antioxidant activity at least for 24 hours and was able to protect human epithelial cells from the detrimental effects of oxidative stress. These results demonstrate that LF-loaded contact lenses could represent a new therapeutic approach to treat ocular surface pathologies characterized by high levels of oxidative stress.

Characterization of a Dihydropyridine‐insensitive Cav1.3 Channel

The L‐type voltage‐gated calcium channels (L‐VGCCs), Cav1.2 and Cav1.3, play an important role in different physiological processes such as action potential firing, exocytosis and muscle contraction. Due to a lack of selectivity for the dihydropyridine (DHP) class of L‐VGCC blockers, a Cav1.3 mutant that is insensitive to DHPs (Cav1.3/DHPi) has been used to dissect the role of Cav1.3 in glucose‐stimulated insulin secretion and other cellular activities. However, the Cav1.3/DHPi mutant channel has not been biophysically characterized. Our lab created the Cav1.3/DHPi by mutating Thr1033 and Gln1037 that are located in segment 5 of domain III (IIIS5) of the rat neuronal Cav1.3 clone (accession no. AF30010.1) to Tyr and Met, respectively. Ba2+ current (IBa) elicited by different voltage steps from tsA cells expressing Cav channels with and without the presence of drugs was measured. The voltage‐dependent activation and inactivation of Cav1.3/DHPi (V1/2 inact.= ‐26.6±1.2mV, V1/2 act.= ‐22.3±1.1mV) resembles that of wild type Cav1.3 (Cav1.3/WT) (V1/2 inact.= ‐36.0±1.3mV, V1/2 act.= ‐29.8±1.5mV). Cav1.3/DHPi is over 100 fold more insensitive to nifedipine compared to Cav1.3/WT (IC50 for Cav1.3/WT= ~0.55μM; IC50 for Cav1.3/DHPi=~70μM). However, the non‐DHP blockers, diltiazem (500μM) and the recently debated Cav1.3 selective blocker, compound 8 (50μM) achieved comparable inhibition of Cav1.3/WT and Cav1.3/DHPi‐mediated current. Our findings showed that Thr1033 and Gln1037 are not only critical, but also specific for DHP block of Cav1.3. The Cav1.3/DHPi mutant is a useful tool to screen for Cav1.3 specific blockers by removing the DHP binding site which is common to both Cav1.2 and Cav1.3.

Radiotherapy induces responsiveness of a resistant mammary carcinoma to PD-1 blockade

ligands, PDL-1/2, were upregulated by RT on TSA cells and tumor-infiltrating myeloid cells, suggesting PD-1’s interaction with its ligands may limit RT-activated antitumor T-cell activity. Consistent with this hypothesis, RMP1-14 alone had no effect on tumor growth; RT delayed growth (p < 0.01), but only 1/6 mice showed tumor regression, whereas all mice receiving RT+RMP114 completely rejected tumors by day 25. In spleen, RMP1-14 had no effect on AH1-specific CD8 + T-cells