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During the past century, from the advent of preclinical modeling to the establishment of clinical trials, the hypothesis that host defenses regulate tumor growth (posited and refined by leaders in the field of cancer immunity) has become accepted as a scientific pillar in oncology. Since the turn of the millennium, a search has been under way for the best therapeutic approach to reprogram the immune system to recognize tumor cells that have undergone "immune escape." This quest has led some to question conventional scientific views of tumor cell kill, including the role of host immunity in patients treated with radiation therapy. In the last two decades, evidence has accumulated that radiation therapy can effectively convert a potentially lethal cancer into an in situ personalized vaccine. Herein, we review the underlying mechanisms and maneuvers responsible for in situ vaccine production.

Longitudinal studies are needed to evaluate the SARS-CoV-2 mRNA vaccine antibody response under real-world conditions. This longitudinal study investigated the quantity and quality of SARS-CoV-2 antibody response in 846 specimens from 350 patients, comparing BNT162b2-vaccinated individuals (19 previously diagnosed with COVID-19, termed RecoVax; and 49 never diagnosed, termed NaiveVax) with 122 hospitalized unvaccinated (HospNoVax) and 160 outpatient unvaccinated (OutPtNoVax) COVID-19 patients. NaiveVax experienced delay in generating SARS-CoV-2 total antibodies (TAb) and surrogate neutralizing antibodies (SNAb) after the first vaccine dose (D1) but rapid increase in antibody levels after the second dose (D2). However, these never reached RecoVax's robust levels. In fact, NaiveVax TAb and SNAb levels decreased 4 weeks after D2. For the most part, RecoVax TAb persisted, after reaching maximal levels 2 weeks after D2, but SNAb decreased significantly about 6 months after D1. Although NaiveVax avidity lagged behind that of RecoVax for most of the follow-up periods, NaiveVax did reach similar avidity by about 6 months after D1. These data suggest that 1 vaccine dose elicits maximal antibody response in RecoVax and may be sufficient. Also, despite decreasing levels in TAb and SNAb over time, long-term avidity may be a measure worth evaluating and possibly correlating to vaccine efficacy.

Longitudinal studies are needed to evaluate the SARS-CoV-2 mRNA vaccine antibody response under “real-world” conditions. This longitudinal study investigated the quantity and quality of SARS-CoV-2 antibody response in 846 specimens from 350 subjects: comparing BNT162b2-vaccinated individuals (19 previously diagnosed with COVID-19 [RecoVax]; 49 never been diagnosed [NaïveVax]) to 122 hospitalized unvaccinated (HospNoVax) and 160 outpatient unvaccinated (OutPtNoVax) COVID-19 patients.NaïveVax experienced a delay in generating SARS-CoV-2 total antibody levels (TAb) and neutralizing antibodies (SNAb) after the 1st vaccine dose (D1), but a rapid increase in antibody levels was observed after the 2nddose (D2). However, these never reached the robust levels observed in RecoVax. In fact, NaïveVax TAb and SNAb levels decreased 4-weeks post-D2 (p=0.003;p

Vaccines are critical for curtailing the COVID-19 pandemic (1, 2). In the USA, two highly protective mRNA vaccines are available: BNT162b2 from Pfizer/BioNTech and mRNA-1273 from Moderna (3, 4). These vaccines induce antibodies to the SARS-CoV-2 S-protein, including neutralizing antibodies (NAbs) predominantly directed against the Receptor Binding Domain (RBD) (1-4). Serum NAbs are induced at modest levels within ∼1 week of the first dose, but their titers are strongly boosted by a second dose at 3 (BNT162b2) or 4 weeks (mRNA-1273) (3, 4). SARS-CoV-2 is most commonly transmitted nasally or orally and infects cells in the mucosae of the respiratory and to some extent also the gastrointestinal tract (5). Although serum NAbs may be a correlate of protection against COVID-19, mucosal antibodies might directly prevent or limit virus acquisition by the nasal, oral and conjunctival routes (5). Whether the mRNA vaccines induce mucosal immunity has not been studied. Here, we report that antibodies to the S-protein and its RBD are present in saliva samples from mRNA-vaccinated healthcare workers (HCW). Within 1-2 weeks after their second dose, 37/37 and 8/8 recipients of the Pfizer and Moderna vaccines, respectively, had S-protein IgG antibodies in their saliva, while IgA was detected in a substantial proportion. These observations may be relevant to vaccine-mediated protection from SARS-CoV-2 infection and disease.

Cells succumbing to stress via regulated cell death (RCD) can initiate an adaptive immune response associated with immunological memory, provided they display sufficient antigenicity and adjuvanticity. Moreover, multiple intracellular and microenvironmental features determine the propensity of RCD to drive adaptive immunity. Here, we provide an updated operational definition of immunogenic cell death (ICD), discuss the key factors that dictate the ability of dying cells to drive an adaptive immune response, summarize experimental assays that are currently available for the assessment of ICD in vitro and in vivo, and formulate guidelines for their interpretation. ispartof: JOURNAL FOR IMMUNOTHERAPY OF CANCER vol:8 issue:1 ispartof: location:England status: published

Focal radiation therapy enhances systemic responses to anti-CTLA-4 antibodies in preclinical studies and in some patients with melanoma1-3, but its efficacy in inducing systemic responses (abscopal responses) against tumors unresponsive to CTLA-4 blockade remained uncertain. Radiation therapy promotes the activation of anti-tumor T cells, an effect dependent on type I interferon induction in the irradiated tumor4-6. The latter is essential for achieving abscopal responses in murine cancers6. The mechanisms underlying abscopal responses in patients treated with radiation therapy and CTLA-4 blockade remain unclear. Here we report that radiation therapy and CTLA-4 blockade induced systemic anti-tumor T cells in chemo-refractory metastatic non-small-cell lung cancer (NSCLC), where anti-CTLA-4 antibodies had failed to demonstrate significant efficacy alone or in combination with chemotherapy7,8. Objective responses were observed in 18% of enrolled patients, and 31% had disease control. Increased serum interferon-β after radiation and early dynamic changes of blood T cell clones were the strongest response predictors, confirming preclinical mechanistic data. Functional analysis in one responding patient showed the rapid in vivo expansion of CD8 T cells recognizing a neoantigen encoded in a gene upregulated by radiation, supporting the hypothesis that one explanation for the abscopal response is radiation-induced exposure of immunogenic mutations to the immune system.

BACKGROUND Stereotactic body radiotherapy (SBRT) for localized prostate cancer has potential advantages over traditional radiotherapies. Herein, the authors compared national trends in use, complications, and costs of SBRT with those of traditional radiotherapies. METHODS The authors identified men who underwent SBRT, intensity-modulated radiotherapy (IMRT), brachytherapy, and proton beam therapy as primary treatment of prostate cancer between 2004 and 2011 from Surveillance, Epidemiology, and End Results Program (SEER)-Medicare linked data. Temporal trend of therapy use was assessed using the Cochran-Armitage test. Two-year outcomes were compared using the chi-square test. Median treatment costs were compared using the Kruskal-Wallis test. RESULTS A total of 542 men received SBRT, 9647 received brachytherapy, 23,408 received IMRT, and 800 men were treated with proton beam therapy. There was a significant increase in the use of SBRT and proton beam therapy (P

Purpose The incidence of brain metastases is increasing as a result of more routine diagnostic imaging and improved extracranial systemic treatment strategies. As noted in recent consensus guidelines, postoperative stereotactic radiosurgery (SRS) to the resection cavity has lower rates of local control than whole brain radiation therapy but improved cognitive outcomes. Further analyses are needed to improve local control and minimize toxicity. Methods and materials Patients receiving SRS to a resection cavity between 2006 and 2016 were retrospectively analyzed. Presurgical variables, including tumor location, diameter, dural/meningeal contact, and histology, were collected, as were SRS treatment parameters. Patients had routine follow-up with magnetic resonance imaging, and those noted to have local failure were further assessed for the recurrence location, distance from the target volume, and dosimetric characteristics. Results Overall, 82 patients and 85 resection cavities underwent postoperative SRS during the study period. Of these, 58 patients with 60 resection cavities with available follow-up magnetic resonance imaging scans were included in this analysis. With a median follow-up of 19.8 months, local recurrence occurred in 12 of the resection cavities for a 15% 1-year and 18% 2-year local recurrence rate. Pretreatment tumor volume contacted the dura/meninges in 100% of cavities with recurrence versus 67% of controlled cavities (P = .025). A total of 5 infield, 5 marginal, and 4 out-of-field recurrences were found, with a median distance to the centroid from the target volume of 3 mm. The addition of a 10-mm dural margin increased the target volume overlap with the recurrence contours for 10 of the 14 recurrences. Conclusions Dural contact was associated with an increased rate of recurrence for patients who received SRS to a surgical cavity, and the median distance of marginal recurrences from the target volume was 3 mm. These results provide evidence in support of recent consensus guidelines suggesting that additional dural margin on SRS volumes may benefit local control.

Advances in understanding the mechanisms that underlie the interplay between radiation-invoked immune responses and tumor regression are underway. Emerging applications of local radiotherapy as an immunologic adjuvant have provided radiation oncologists with a method for converting malignant cells into endogenous anticancer vaccines. The dispersion of radiotherapy-induced immune-stimulating tumor antigens released from dying tumor cells into the surrounding milieu (known as immunogenic cell death, Fig. 1), is one such exploitable process that contributes to the propagation of antitumor immunity. Downstream components of the immune system may suppress, promote, or ambiguously affect antitumoral responses. Additionally, host, tumor, and treatment-related characteristics govern the significance of these signals, thereby dictating therapeutic outcomes. Herein, we review the process of radiotherapy-induced immunogenic cell death and its role in generating an in situ vaccine to help refine radioimmunotherapy-based protocols.

In parallel with the discovery of the taxanes, our understanding of the molecular underpinnings that comprise the classic biologic principles of fractionated radiotherapy has rapidly evolved over the past half century. Early studies have implicated DNA as the primary target for radiation-induced lethality. More recently, however, the molecular biology involved in radiosensitization of tumor cells has been unveiled. Specifically, factors associated with DNA damage and cell killing, collectively known as the 'four Rs' of radiobiology, including (r)eassortment of tumor cells into the radiosensitive phases of the cell cycle (G2/M), (r)eoxygenation of hypoxic areas within a tumor, (r)epair of sublethal DNA damage, and (r)epopulation of surviving tumor cells, have been elucidated, and upon manipulation of each factor or a combination of factors a significant impact on radiation-associated tumor control probabilities was found. Not only does spatial cooperation have a theoretical benefit in patients with undetectable micrometastatic disease at presentation, but the manipulation of either of the 'four Rs' using taxanes provokes further local radiation-associated tumor cell killing with an associated improvement in clinical responses. Numerous studies have shown that taxanes radiosensitize tumor cells directly and/or indirectly by perturbing the tumor microenvironment in a time-dependent and dose-dependent manner. Herein, the impact of taxanes on radiobiological tenets as a mode of radiosensitizing tumor cells and their clinical implications are reviewed.

A posteriori evidence suggests that radiotherapy to a targeted tumor can elicit an immune-mediated abscopal (ab-scopus, away from the target) effect in nontargeted tumors, when combined with an anti-CTL antigen-4 (CTLA-4) monoclonal antibody. Concurrent radiotherapy and CTLA-4 blockade induced immune-mediated abscopal effects in poorly immunogenic preclinical tumor models and patients with metastatic melanoma. However, no such reports exist for patients with metastatic lung adenocarcinoma. We report the first abscopal response in a treatment-refractory lung cancer patient treated with radiotherapy and ipilimumab (a human anti-CTLA-4 monoclonal antibody). A posttreatment increase in tumor-infiltrating cytotoxic lymphocytes, tumor regression, and normalization of tumor markers was observed. One year after treatment with concurrent radiotherapy and ipilimumab, the patient is without evidence of disease. Cancer Immunol Res; 1(6); 365–72. ©2013 AACR.

The effects of ionizing radiation on the immune system are under active preclinical and clinical investigation. In the United States, approximately 50 clinical trials are currently testing the addition of radiation therapy to various immunotherapeutic strategies. With such an exponential growth of interest, it might be useful to pause and reflect on the challenges associatedwith thepromise of this newareaof research. The recently reported results by Kwon et al (1) of a large, multi-institutional, prospective, randomized trial in men with metastatic castrate-resistant and docetaxelrefractory prostate cancer offer the occasion for this editorial. All patients in this study were pretreated with radiation therapy targeting bone metastases (8 Gy in a single fraction) and then randomly assigned to receive either ipilimumab or placebo. The rationale for the experimental arm was based on the hypothesis that ipilimumab would enable antitumor immunity, after radiation had converted the tumor into an in situ vaccine by inducing immunogenic cell death and exposing cryptic tumor antigens (1-3). Unfortunately, this important and wellconducted study failed to meet its primary endpoint of an improved median overall survival for the experimental arm. Only on subset analysis was it possible to detect a difference in median overall survival for patients with “good prognostic features” (1). Though not yet reported by Kwon et al, immunologic surrogates for antitumor immunity (such as changes in the peripheral blood immune cells, tumor

The immune system plays an important role in both counteracting and facilitating cancer development, as well as contributing to the effects of standard cancer treatments. As such, the immune system is most certainly involved in the response of glioblastoma to radiotherapy; however, it is difficult to elucidate the specific role of immunity due to limitations in animal models and the difficulty in obtaining tissue to study molecular correlates of progression and response. Yet there is abundant evidence that leukocytes and other immune mediators recognize and respond to glioma cells, and indirect evidence that they participate in the therapeutic response to radiation. After a brief introduction on the role of the innate and adaptive immune response to cancer, we review the effects of radiotherapy on the anti-tumor immune response, including inflammation, T-cell priming, and immune suppression. We first introduce the concept of radiation induced immunogenic cell death, its application to the unique immunological landscape of the CNS, and present available evidence that radiation induces an immune response against glioblastoma. We then discuss some of the barriers of the brain/tumor microenvironment that may interfere with effective anti-tumor immunity, and conclude with suggested approaches to better harness the immune response in the treatment of glioblastoma with radiotherapy.

The alkylating agent temozolomide, in combination with surgery and radiation, is the current standard of care for patients with glioblastoma. However, despite this extensive therapeutic effort, the inclusion of temozolomide extends survival only by a few short months. Among the factors contributing to chemoresistance is elevated expression of the endoplasmic reticulum (ER) chaperone GRP78 (glucose-regulated protein 78; BiP), a key pro-survival component of the ER stress response system. Because the green tea component EGCG (epigallocatechin 3-gallate) had been shown to inhibit GRP78 function, we investigated whether this polyphenolic agent would be able to increase the therapeutic efficacy of temozolomide in preclinical models of glioblastoma. Mice with intracranially implanted human U87 (p53 wild type) or U251 (p53 mutant) glioblastoma cells were treated with temozolomide and EGCG, alone and in combination. We found that EGCG alone did not provide survival benefit, but significantly improved the existing therapeutic effect of temozolomide, i.e., life extension was substantially greater under combination therapy as compared to temozolomide therapy alone. Immunohistochemical analysis of tumor tissue revealed increased expression levels of GRP78 in temozolomide-treated animals, which was diminished when temozolomide was combined with EGCG. Parallel in vitro experiments with siRNA targeting GRP78 or its major pro-apoptotic antagonist CHOP (CCAAT/enhancer binding protein homologous protein/GADD153) further established a critical role of the ER stress response system, where si-GRP78 sensitized cells to treatment with temozolomide, and si-CHOP provided protection from drug-induced toxicity. Thus, ER stress-regulatory components affect the chemotherapeutic response of glioblastoma cells to treatment with temozolomide, and inclusion of EGCG is able to increase the therapeutic efficacy of this DNA-damaging agent.

Our laboratory has previously shown that a novel compound, 2,5-dimethyl-celecoxib (DMC), which is structurally similar to the cyclooxygenase-2 (COX-2) inhibitor celecoxib but lacks the COX-2–inhibitory function, mimics the antitumor effects of celecoxib. Most studies on DMC, however, focused on its effects on tumor cells. Here, we investigated the activities of DMC as an antiangiogenic agent in both in vitro and in vivo systems. Using primary cultures of human glioma specimens, we found that DMC treatment was cytotoxic to tumor-associated brain endothelial cells (TuBEC), which was mediated through the endoplasmic reticulum stress pathway. In contrast, confluent cultures of quiescent human BEC did not undergo cell death. DMC potently suppressed the proliferation and migration of the TuBEC. DMC caused no apparent effects on the secretion of vascular endothelial growth factor and interleukin-8 but inhibited the secretion of endothelin-1 in tumor-associated EC. DMC treatment of glioma xenografts in mice resulted in smaller tumors with a pronounced reduction in microvessel density compared with untreated mice. In vitro and in vivo analyses confirmed that DMC has antivascular activity. Considering that DMC targets both tumor cells and tumor-associated ECs, this agent is a promising anticancer drug. Mol Cancer Ther; 9(3); 631–41

Temozolomide is considered the standard of care and drug of choice for the treatment of initially diagnosed malignant gliomas. Although well tolerated, temozolomide still has limited clinical efficacy. Following drug treatment, patient prognosis still remains poor; tumor recurrence is almost universal. We hypothesized that this lack of effectiveness with temozolomide is because this drug does not target the glioma microenvironment, which is highly vascular in malignant gliomas. To test this hypothesis we analyzed the effects of temozolomide on the tumor vasculature in vitro and in vivo. We found that this drug did not affect the viability or proliferation rate of endothelial cells isolated from human glioma specimens, although temozolomide was highly cytotoxic to the glioma cell lines U87MG and U251. Furthermore, temozolomide did not inhibit the migration of these glioma-associated endothelial cells, a key mechanism responsible for tumor angiogenesis. In in vivo studies, using the intracranial glioma mouse model, temozolomide did not cause a pronounced effect on microvessel density. Our findings show that temozolomide has no apparent effect on the glioma vascular microenvironment. Thus combination therapy with anti-vascular agents may enhance temozolomide effectiveness as glioma therapeutic protocol.

The anticancer potency of green tea and its individual components is being intensely investigated, and some cancer patients already self-medicate with this “miracle herb” in hopes of augmenting the anticancer outcome of their chemotherapy. Bortezomib (BZM) is a proteasome inhibitor in clinical use for multiple myeloma. Here, we investigated whether the combination of these compounds would yield increased antitumor efficacy in multiple myeloma and glioblastoma cell lines in vitro and in vivo. Unexpectedly, we discovered that various green tea constituents, in particular (-)-epigallocatechin gallate (EGCG) and other polyphenols with 1,2-benzenediol moieties, effectively prevented tumor cell death induced by BZM in vitro and in vivo. This pronounced antagonistic function of EGCG was evident only with boronic acid–based proteasome inhibitors (BZM, MG-262, PS-IX), but not with several non–boronic acid proteasome inhibitors (MG-132, PS-I, nelfinavir). EGCG directly reacted with BZM and blocked its proteasome inhibitory function; as a consequence, BZM could not trigger endoplasmic reticulum stress or caspase-7 activation, and did not induce tumor cell death. Taken together, our results indicate that green tea polyphenols may have the potential to negate the therapeutic efficacy of BZM and suggest that consumption of green tea products may be contraindicated during cancer therapy with BZM.

The proteasome inhibitor bortezomib (Velcade) is known to trigger endoplasmic reticulum (ER) stress via the accumulation of obsolete and damaged proteins. The selective cyclooxygenase-2 (COX-2) inhibitor celecoxib (Celebrex) causes ER stress through a different mechanism (i.e., by causing leakage of calcium from the ER into the cytosol). Each of these two mechanisms has been implicated in the anticancer effects of the respective drug. We therefore investigated whether the combination of these two drugs would lead to further increased ER stress and would enhance their antitumor efficacy. With the use of human glioblastoma cell lines, we show that this is indeed the case. When combined, bortezomib and celecoxib triggered elevated expression of the ER stress markers GRP78/BiP and CHOP/GADD153, caused activation of c-Jun NH2-terminal kinase and ER stress-associated caspase-4, and greatly increased apoptotic cell death. Small interfering RNA–mediated knockdown of the protective ER chaperone GRP78/BiP further sensitized the tumor cells to killing by the drug combination. The contribution of celecoxib was independent of the inhibition of COX-2 because a non-coxib analogue of this drug, 2,5-dimethyl-celecoxib (DMC), faithfully and more potently mimicked these combination effects in vitro and in vivo. Taken together, our results show that combining bortezomib with celecoxib or DMC very potently triggers the ER stress response and results in greatly increased glioblastoma cytotoxicity. We propose that this novel drug combination should receive further evaluation as a potentially effective anticancer therapy. [Cancer Res 2008;68(3):843–51]

International audience; The efficacy of the anti-cancer immunomodulatory agent cyclophosphamide (CTX) relies on intestinal bacteria. How and which relevant bacterial species are involved in tumor immunosurveillance, and their mechanism of action are unclear. Here, we identified two bacterial species, Enterococcus hirae and Barnesiella intestinihominis that are involved during CTX therapy. Whereas E. hirae translocated from the small intestine to secondary lymphoid organs and increased the intratumoral CD8/ Treg ratio, B. intestinihominis accumulated in the colon and promoted the infiltration of IFN-gamma-producing gamma delta Tau cells in cancer lesions. The immune sensor, NOD2, limited CTX-induced cancer immunosurveillance and the bioactivity of these microbes. Finally, E. hirae and B. intestinihominis specific-memory Th1 cell immune responses selectively predicted longer progression-free survival in advanced lung and ovarian cancer patients treated with chemo-immunotherapy. Altogether, E. hirae and B. intestinihominis represent valuable ''oncomicrobiotics'' ameliorating the efficacy of the most common alkylating immunomodulatory compound.

Summary Background An abscopal response describes radiotherapy-induced immune-mediated tumour regression at sites distant to the irradiated field. Granulocyte-macrophage colony-stimulating factor is a potent stimulator of dendritic cell maturation. We postulated that the exploitation of the pro-immunogenic effects of radiotherapy with granulocyte-macrophage colony-stimulating factor might result in abscopal responses among patients with metastatic cancer. Methods Patients with stable or progressing metastatic solid tumours, on single-agent chemotherapy or hormonal therapy, with at least three distinct measurable sites of disease, were treated with concurrent radiotherapy (35 Gy in ten fractions, over 2 weeks) to one metastatic site and granulocyte-macrophage colony-stimulating factor (125 μg/m 2 subcutaneously injected daily for 2 weeks, starting during the second week of radiotherapy). This course was repeated, targeting a second metastatic site. A Simon's optimal two-stage design was chosen for this trial: an additional 19 patients could be enrolled in stage 2 only if at least one patient among the first ten had an abscopal response. If no abscopal responses were seen among the first ten patients, the study would be deemed futile and terminated. The primary endpoint was the proportion of patients with an abscopal response (defined as at least a 30% decrease in the longest diameter of the best responding abscopal lesion). Secondary endpoints were safety and survival. Analyses were done based on intention to treat. The trial has concluded accrual, and is registered with ClinicalTrials.gov, number NCT02474186. Findings From April 7, 2003, to April 3, 2012, 41 patients with metastatic cancer were enrolled. In stage 1 of the Simon's two-stage design, ten patients were enrolled: four of the first ten patients had abscopal responses. Thus, the trial proceeded to stage 2, as planned, and an additional 19 patients were enrolled. Due to protocol amendments 12 further patients were enrolled. Abscopal responses occurred in eight (27·6%, 95% CI 12·7–47·2) of the first 29 patients, and 11 (26·8%, 95% CI 14·2–42·9) of 41 accrued patients (specifically in four patients with non-small-cell lung cancer, five with breast cancer, and two with thymic cancer). The most common grade 3–4 adverse events were fatigue (six patients) and haematological (ten patients). Additionally, a serious adverse event of grade 4 pulmonary embolism occurred in one patient. Interpretation The combination of radiotherapy with granulocyte-macrophage colony-stimulating factor produced objective abscopal responses in some patients with metastatic solid tumours. This finding represents a promising approach to establish an in-situ anti-tumour vaccine. Further research is warranted in this area. Funding New York University School of Medicine's Department of Radiation Oncology and Cancer Institute.

OBJECT Chloroquine (CQ) is a quinoline-based drug widely used for the prevention and treatment of malaria. More recent studies have provided evidence that this drug may also harbor antitumor properties, whereby CQ possesses the ability to accumulate in lysosomes and blocks the cellular process of autophagy. Therefore, the authors of this study set out to investigate whether CQ analogs, in particular clinically established antimalaria drugs, would also be able to exert antitumor properties, with a specific focus on glioma cells. METHODS Toward this goal, the authors treated different glioma cell lines with quinine (QN), quinacrine (QNX), mefloquine (MFQ), and hydroxychloroquine (HCQ) and investigated endoplasmic reticulum (ER) stress–induced cell death, autophagy, and cell death. RESULTS All agents blocked cellular autophagy and exerted cytotoxic effects on drug-sensitive and drug-resistant glioma cells with varying degrees of potency (QNX > MFQ > HCQ > CQ > QN). Furthermore, all quinoline-based drugs killed glioma cells that were highly resistant to temozolomide (TMZ), the current standard of care for patients with glioma. The cytotoxic mechanism involved the induction of apoptosis and ER stress, as indicated by poly(ADP-ribose) polymerase (PARP) cleavage and CHOP/GADD153. The induction of ER stress and resulting apoptosis could be confirmed in the in vivo setting, in which tumor tissues from animals treated with quinoline-based drugs showed increased expression of CHOP/GADD153, along with elevated TUNEL staining, a measure of apoptosis. CONCLUSIONS Thus, the antimalarial compounds investigated in this study hold promise as a novel class of autophagy inhibitors for the treatment of newly diagnosed TMZ-sensitive and recurrent TMZ-resistant gliomas.

OBJECT Bevacizumab (Avastin), an antibody to vascular endothelial growth factor (VEGF), alone or in combination with irinotecan (Camptosar [CPT-11]), is a promising treatment for recurrent glioblastoma. However, the intravenous (IV) administration of bevacizumab produces a number of systemic side effects, and the increase in survival it provides for patients with recurrent glioblastoma is still only a few months. Because bevacizumab is an antibody against VEGF, which is secreted into the extracellular milieu by glioma cells, the authors hypothesized that direct chronic intratumoral delivery techniques (i.e., convection-enhanced delivery [CED]) can be more effective than IV administration. To test this hypothesis, the authors compared outcomes for these routes of bevacizumab application with respect to animal survival, microvessel density (MVD), and inflammatory cell distribution. METHODS Two human glioma cell lines, U87 and U251, were used as sources of intracranial tumor cells. The glioma cell lines were implanted into the brains of mice in an orthotopic xenograft mouse tumor model. After 7 days, the mice were treated with one of the following: 1) vehicle, 2) CED bevacizumab, 3) IV bevacizumab, 4) intraperitoneal (IP) irinotecan, 5) CED bevacizumab plus IP irinotecan, or 6) IV bevacizumab plus IP irinotecan. Alzet micro-osmotic pumps were used to introduce bevacizumab directly into the tumor. Survival was monitored. Excised tumor tissue samples were immunostained to measure MVD and inflammatory cell and growth factor levels. RESULTS The results demonstrate that mice treated with CED of bevacizumab alone or in combination with irinotecan survived longer than those treated systemically; CED-treated animals survived 30% longer than IV-treated animals. In combination studies, CED bevacizumab plus CPT-11 increased survival by more than 90%, whereas IV bevacizumab plus CPT-11 increased survival by 40%. Furthermore, CED bevacizumab-treated tissues exhibited decreased MVD compared with that of IV-treated tissues. In additional studies, the infiltration of macrophages and dendritic cells into CED-treated animals were increased compared with those in IV-treated animals, suggesting a highly active inflammatory response taking place in CED-treated mice. CONCLUSIONS The administration of bevacizumab via CED increases survival over that of treatment with IV bevacizumab. Thus, CED of bevacizumab alone or in combination with chemotherapy can be an effective protocol for treating gliomas.

International audience; Antibodies targeting CTLA-4 have been successfully used as cancer immunotherapy. We find that the antitumor effects of CTLA-4 blockade depend on distinct Bacteroides species. In mice and patients, Tcell responses specific for B. thetaiotaomicron or B. fragilis were associated with the efficacy of CTLA-4 blockade. Tumors in antibiotic-treated or germ-free mice did not respond to CTLA blockade. This defect was overcome by gavage with B. fragilis, by immunization with B. fragilis polysaccharides, or by adoptive transfer of B. fragilis-specific T cells. Fecal microbial transplantation from humans to mice confirmed that treatment of melanoma patients with antibodies against CTLA-4 favored the outgrowth of B. fragilis with anticancer properties. This study reveals a key role for Bacteroidales in the immunostimulatory effects of CTLA-4 blockade.

Object In a recent clinical trial, patients with newly diagnosed glioblastoma multiforme benefited from chloroquine (CQ) in combination with conventional therapy (resection, temozolomide [TMZ], and radiation therapy). In the present study, the authors report the mechanism by which CQ enhances the therapeutic efficacy of TMZ to aid future studies aimed at improving this therapeutic regimen. Methods Using in vitro and in vivo experiments, the authors determined the mechanism by which CQ enhances TMZ cytotoxicity. They focused on the inhibition-of-autophagy mechanism of CQ by knockdown of the autophagy-associated proteins or treatment with autophagy inhibitors. This mechanism was tested using an in vivo model with subcutaneously implanted U87MG tumors from mice treated with CQ in combination with TMZ. Results Knockdown of the autophagy-associated proteins (GRP78 and Beclin) or treatment with the autophagy inhibitor, 3-methyl adenine (3-MA), blocked autophagosome formation and reduced CQ cytotoxicity, suggesting that autophagosome accumulation precedes CQ-induced cell death. In contrast, blocking autophagosome formation with knockdown of GRP78 or treatment with 3-MA enhanced TMZ cytotoxicity, suggesting that the autophagy pathway protects from TMZ-induced cytotoxicity. CQ in combination with TMZ significantly increased the amounts of LC3B-II (a marker for autophagosome levels), CHOP/GADD-153, and cleaved PARP (a marker for apoptosis) over those with untreated or individual drug-treated glioma cells. These molecular mechanisms seemed to take place in vivo as well. Subcutaneously implanted U87MG tumors from mice treated with CQ in combination with TMZ displayed higher levels of CHOP/GADD-153 than did untreated or individual drug-treated tumors. Conclusions Taken together, these results demonstrate that CQ blocks autophagy and triggers endoplasmic reticulum stress, thereby increasing the chemosensitivity of glioma cells to TMZ.

The main role of the immune system is to restore tissue homeostasis when altered by pathogenic processes, including neoplastic transformation. Immune-mediated tumor rejection has been recognized as an extrinsic tumor suppressor mechanism that tumors need to overcome to progress. By the time a tumor becomes clinically apparent it has successfully escaped immune control by establishing an immunosuppressive microenvironment. Ionizing radiation applied locally to a tumor alters these tumor-host interactions. Accumulating evidence indicates that standard therapeutic doses of radiation have the potential to recover tumor immunogenicity and convert the tumor into an in situ personalized vaccine. Radiotherapy induces an immunogenic tumor cell death promoting cross-presentation of tumor-derived antigens by dendritic cells to T cells. In addition, radiotherapy stimulates chemokine-mediated recruitment of effector T cells to the tumor, and cellular recognition and killing by T cells that is facilitated by upregulation of major histocompatibility antigens, NKG2D ligands, adhesion molecules and death receptors. Despite these effects, radiotherapy alone is only rarely capable of generating enough proinflammatory signals to sufficiently overcome suppression, as it can also activate immunosuppressive factors. However, our group and others have shown that when combined with targeted immunotherapy agents radiotherapy significantly contributes to a therapeutically effective anti-tumor immune response. To illustrate this partnership between radiation and immunotherapy we will discuss as an example our experience in preclinical models and the molecular mechanisms identified. Additionally, the clinical translation of these combinations will be discussed.

Established tumors are typified by an immunosuppresive microenvironment. Countering this naturally occurring phenomenon, emerging evidence suggests that radiation promotes a proimmunogenic milieu within the tumor capable of stimulating host cancer-specific immune responses. Three cryptic immunogenic components of cytotoxic-agent induced cell death—namely, calreticulin cell surface exposure, the release of high mobility group box 1 (HMGB1) protein, and the liberation of ATP—have been previously shown to be critical for dendritic cell (DC) activation and effector T-cell priming. Thus, these immune-mobilizing components commonly presage tumor rejection in response to treatment. We initially set out to address the hypothesis that radiation-induced immunogenic cell death (ICD) is dose-dependent. Next, we hypothesized that radiation would enhance chemotherapy-induced ICD when given concomitantly, as suggested by the favorable clinical outcomes observed in response to analogous concurrent chemoradiation regimens. Thus, we designed an in vitro assay to examine the 3 hallmark features of ICD at clinically relevant doses of radiation. We then tested the immunogenic-death inducing effects of radiation combined with carboplatin or paclitaxel, focusing on these combinations to mimic chemoradiation regimens actually used in clinical trials of early stage triple negative [NCT0128953/NYU-10–01969] and locally advanced [NYU-06209] breast cancer patients, respectively. Despite the obvious limitations of an in vitro model, radiotherapy produced both a dose-dependent induction and chemotherapeutic enhancement of ICD. These findings provide preliminary evidence that ICD stimulated by either high-dose radiotherapy alone, or concurrent chemoradiation regimens, may contribute to the establishment of a peritumoral proimmunogenic milieu.

Purpose: To determine feasibility and explore the clinical efficacy of concurrent radiotherapy and carboplatin as adjuvant treatment of triple negative breast cancer (TNBC). Patients and Methods: Women with Stage I-II TNBC were treated after surgery in a phase I–II prospective trial [NCT01289353]. Weekly carboplatin (AUC = 2.0) was delivered for 6 weeks. Concurrent radiotherapy was delivered in the prone position during weeks 2–4, for a total dose of 40.5 Gy in 15 fractions to the breast, and 46.5 Gy in 17 fractions to the tumor bed. Adverse events (AE) were assessed weekly during treatment, once at 45–60 d, and every 6 mo thereafter, using the Common Terminology Criteria for AE (CTCAE) v3.0. Results: A total of 39 patients accrued and 36 received treatment. Eight patients (22%, exact 95% CI: 10%, 39%) developed grade 2 or greater acute radiation dermatitis. Overall, grade 2 AE were seen in nine and grade 3 in two patients. Twenty-three patients (64%) received additional adjuvant chemotherapy. With a median follow-up of 48 mo, 34/36 (94%) are alive and disease free. One patient died of pulmonary failure with possible but unproven breast cancer recurrence, and one patient died of pelvic malignancy. One patient recurred locally and is alive and disease free after surgical management. Brisk lymphocytic infiltrate was present pre-treatment in 39% of 18 patients with evaluable tumor. Conclusions: Adjuvant concurrent carboplatin and prone accelerated radiotherapy is a well-tolerated and promising treatment of early stage TNBC. The observed 3% compares favorably with the expected 30% recurrence rate within 1–4 y from treatment, warranting further studies.

Background: SD-101 is a CpG oligonucleotide-based TLR9 agonist that engages its target within early and late endosomes of plasmacytoid dendritic cells (pDCs), resulting in DC activation, robust IFN-α responses, and priming of tumor-specific T-cells. We have previously shown that local radiotherapy (RT) may also act as an immune adjuvant and thereby contribute to immune-mediated tumor rejection (J Natl Cancer Inst, 2013: 105:256-265). We hypothesized that the effects of SD-101 may be potentiated by RT and trigger immune-mediated abscopal responses, outside the irradiated field (Int. J. Radiat. Oncol. Biol. Phys., 58:862-870). Methods: BALB/c mice were inoculated s.c. with syngeneic TSA mammary tumor cells in 2 sites (primary [ipsilateral flank] and secondary [contralateral flank]) to detect abscopal responses. When tumors reached an average of 5 mm in diameter, mice were randomly assigned to 1 out of 4 treatment groups: control (untreated), SD-101, RT, or SD-101+RT. RT was delivered to primary tumors (8 Gy x3 fractions) in 3 consecutive days; secondary tumors were completely shielded from RT. SD-101 was injected into primary tumors (50 μg) three times per week starting on the last day of RT. Mice were followed for tumor growth in both the primary and secondary tumors. In a parallel experiment, mice were euthanized 10 days after initiation of therapy for immune analysis. Results: Significant growth delays (p Conclusions: Our studies support the rational use of RT as a powerful therapeutic partner to potentiate the anti-tumor effects of SD-101. Citation Format: Karsten Pilones, Jeffrey Kraynak, Sandra Demaria, Encouse Golden, Silvia Formenti. Radiation potentiates systemic anti-tumor immunity unleashed by a novel TLR9 agonist (SD-101). [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 4985.

Ionizing radiation contributes key danger signals within the tumor microenvironment that are sensed by the immune system. When combined with immunotherapy it can act to successfully vaccinate the carrier against the irradiated tumor. Recruitment and activation of dendritic cells are key components involved in antigen cross presentation and successful vaccination. The clinical translation of preclinical evidence of combining radiation with dendritic cell growth factors confirms their important role for successful, individualized vaccination.

Ionizing radiation (IR) triggers programmed cell death in tumor cells through a variety of highly regulated processes. Radiation-induced tumor cell death has been studied extensively in vitro and is widely attributed to multiple distinct mechanisms, including apoptosis, necrosis, mitotic catastrophe (MC), autophagy, and senescence, which may occur concurrently. When considering tumor cell death in the context of an organism, an emerging body of evidence suggests there is a reciprocal relationship in which radiation stimulates the immune system, which in turn contributes to tumor cell kill. As a result, traditional measurements of radiation-induced tumor cell death, in vitro, fail to represent the extent of clinically observed responses, including reductions in loco-regional failure rates and improvements in metastases free and overall survival. Hence, understanding the immunological responses to the type of radiation-induced cell death is critical. In this review, the mechanisms of radiation-induced tumor cell death are described, with particular focus on immunogenic cell death (ICD). Strategies combining radiotherapy with specific chemotherapies or immunotherapies capable of inducing a repertoire of cancer specific immunogens might potentiate tumor control not only by enhancing cell kill but also through the induction of a successful anti-tumor vaccination that improves patient survival.

Autophagy has been recognized as a potential target for cancer therapy. The antimalarial drug chloroquine (CQ) is able to inhibit autophagy and therefore is being considered for cancer therapeutics. However, the relatively low potency of CQ prompted us to investigate whether other lysosomotropic agents might be more effective, and thus potentially more useful. We therefore compared the cytotoxic efficacy of CQ, the quinoline analog mefloquine (MQ), and the fluoroquinolones ciprofloxacin and levofloxacin in several human breast cancer cell lines. We found that MQ was the most potent compound tested; it inhibited autophagy, triggered endoplasmic reticulum stress, and caused cell death in T47D and MDA-MB-231. Altogether, our study demonstrates superior potency of MQ over CQ and the ability of MQ to produce anticancer effects in both hormone receptor positive and negative breast cancer cell lines, suggesting its usefulness in treating various types of cancer.

The cellular processes of autophagy and endoplasmic reticulum stress (ERS) appear to be interconnected, and it has been proposed that autophagy may serve to reduce ERS via removal of terminally misfolded and aggregated proteins. Conversely, there are indications that blockage of autophagy may increase ERS. Based on earlier work demonstrating that pharmacologically aggravated ERS can result in tumor cell killing, we investigated whether blockage of autophagy would enhance this effect in a therapeutically useful manner. We therefore combined chloroquine (CQ), a pharmacological inhibitor of autophagy, with other drugs known to act as ERS aggravators (ERSA), namely nelfinavir (an HIV protease inhibitor) and celecoxib (a cyclooxygenase-2 inhibitor) or its non-coxib analog 2,5-dimethyl-celecoxib (DMC), and investigated combination drug effects in a variety of breast cancer cell lines. We found that the addition of CQ resulted in synergistic enhancement of tumor cell killing by ERSA compounds, particularly in triple-negative breast cancer (TNBC) cells. This combination effect could also be confirmed in an in vivo model, where CQ boosted low-dose ERSA effects, resulting in rapid deterioration of xenografted tumors in mice. Altogether, our results indicate that combinations of an autophagy inhibitor with pharmacological ERSA (i.e. compounds that lead to ER stress aggravation) should be further explored for potential therapy of otherwise difficult-to-treat TNBC.

The recent success of cancer immunotherapy has demonstrated the power of the immune system to clear tumors, generating renewed enthusiasm for identifying ways to induce antitumor immune responses in patients. Natural antitumor immune responses are detectable in a fraction of patients across multiple malignant neoplasms and can be reactivated by targeting rate-limiting immunosuppressive mechanisms. In most patients, however, interventions to induce a de novo antitumor immune response are necessary. We review growing evidence that radiation therapy targeted to the tumor can convert it into an in situ tumor vaccine by inducing release of antigens during cancer cell death in association with proinflammatory signals that trigger the innate immune system to activate tumor-specific T cells. In addition, radiation’s effects on the tumor microenvironment enhance infiltration of activated T cells and can overcome some of the barriers to tumor rejection. Thus, the complementary effects of radiation on priming and effector phases of antitumor immunity make it an appealing strategy to generate immunity against a patient’s own individual tumor, that through immunological memory, can result in long-lasting systemic responses. Several anecdotal cases have demonstrated the efficacy of combining radiation with available immunotherapies, and results of prospective trials are forthcoming.