Pathogenic variants in autism gene KATNAL2 cause hydrocephalus and disrupt neuronal connectivity by impairing ciliary microtubule dynamics.

Enlargement of the cerebrospinal fluid (CSF)-filled brain ventricles (cerebral ventriculomegaly), the cardinal feature of congenital hydrocephalus (CH), is increasingly recognized among patients with autism spectrum disorders (ASD). KATNAL2, a member of Katanin family microtubule-severing ATPases, is a known ASD risk gene, but its roles in human brain development remain unclear. Here, we show that nonsense truncation of Katnal2 (Katnal2Δ17) in mice results in classic ciliopathy phenotypes, including impaired spermatogenesis and cerebral ventriculomegaly. In both humans and mice, KATNAL2 is highly expressed in ciliated radial glia of the fetal ventricular-subventricular zone as well as in their postnatal ependymal and neuronal progeny. The ventriculomegaly observed in Katnal2Δ17 mice is associated with disrupted primary cilia and ependymal planar cell polarity that results in impaired cilia-generated CSF flow. Further, prefrontal pyramidal neurons in ventriculomegalic Katnal2Δ17 mice exhibit decreased excitatory drive and reduced high-frequency firing. Consistent with these findings in mice, we identified rare, damaging heterozygous germline variants in KATNAL2 in five unrelated patients with neurosurgically treated CH and comorbid ASD or other neurodevelopmental disorders. Mice engineered with the orthologous ASD-associated KATNAL2 F244L missense variant recapitulated the ventriculomegaly found in human patients. Together, these data suggest KATNAL2 pathogenic variants alter intraventricular CSF homeostasis and parenchymal neuronal connectivity by disrupting microtubule dynamics in fetal radial glia and their postnatal ependymal and neuronal descendants. The results identify a molecular mechanism underlying the development of ventriculomegaly in a genetic subset of patients with ASD and may explain persistence of neurodevelopmental phenotypes in some patients with CH despite neurosurgical CSF shunting.

Development of a novel multi­epitope vaccine against the pathogenic human polyomavirus V6/7 using reverse vaccinology.

BACKGROUND: Human polyomaviruses contribute to human oncogenesis through persistent infections, but currently there is no effective preventive measure against the malignancies caused by this virus. Therefore, the development of a safe and effective vaccine against HPyV is of high priority. METHODS: First, the proteomes of 2 polyomavirus species (HPyV6 and HPyV7) were downloaded from the NCBI database for the selection of the target proteins. The epitope identification process focused on selecting proteins that were crucial, associated with virulence, present on the surface, antigenic, non-toxic, and non-homologous with the human proteome. Then, the immunoinformatic methods were used to identify cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes from the target antigens, which could be used to create epitope-based vaccine. The physicochemical features of the designed vaccine were predicted through various online servers. The binding pattern and stability between the vaccine candidate and Toll-like receptors were analyzed through molecular docking and molecular dynamics (MD) simulation, while the immunogenicity of the designed vaccines was assessed using immune simulation. RESULTS: Online tools were utilized to forecast the most optimal epitope from the immunogenic targets, including LTAg, VP1, and VP1 antigens of HPyV6 and HPyV7. A multi-epitope vaccine was developed by combining 10 CTL, 7 HTL, and 6 LBL epitopes with suitable linkers and adjuvant. The vaccine displayed 98.35% of the world's population coverage. The 3D model of the vaccine structure revealed that the majority of residues (87.7%) were located in favored regions of the Ramachandran plot. The evaluation of molecular docking and MD simulation revealed that the constructed vaccine exhibits a strong binding (-1414.0 kcal/mol) towards the host's TLR4. Moreover, the vaccine-TLR complexes remained stable throughout the dynamic conditions present in the natural environment. The immune simulation results demonstrated that the vaccine design had the capacity to elicit robust immune responses in the host. CONCLUSION: The multi-parametric analysis revealed that the designed vaccine is capable of inducing sustained immunity against the selected polyomaviruses, although further in-vivo investigations are needed to verify its effectiveness.

Multifunctional plant virus nanoparticles in the next generation of cancer immunotherapies.

Plant virus nanoparticles (PVNPs) have inherent immune stimulatory ability, and have been investigated as immune adjuvants to stimulate an anti-tumor immune response. The combination of immune stimulation, nanoparticle structure and the ability to deliver other therapeutic molecules provides a flexible platform for cancer immunotherapy. Researching multifunctional PVNPs and their modification will generate novel reagents for cancer immunotherapy. Here we review the properties of PVNPs, and their potential for clinical utilization to activate anti-tumor innate and lymphoid immune responses. PVNPs have potential utility for cancer immunotherapy as vaccine adjuvant, and delivery systems for other reagents as mono immunotherapy or combined with other immunotherapies. This review outlines the potential and challenges in developing PVNPs as cancer immunotherapy reagents.

The Potency of Cowpea Mosaic Virus Particles for Cancer In Situ Vaccination Is Unaffected by the Specific Encapsidated Viral RNA.

Plant virus nanoparticles can be used as drug carriers, imaging reagents, vaccine carriers, and immune adjuvants in the formulation of intratumoral in situ cancer vaccines. One example is the cowpea mosaic virus (CPMV), a nonenveloped virus with a bipartite positive-strand RNA genome with each RNA packaged separately into identical protein capsids. Based on differences in their densities, the components carrying RNA-1 (6 kb) denoted as the bottom (B) component or carrying RNA-2 (3.5 kb) denoted as the middle (M) component can be separated from each other and from a top (T) component, which is devoid of any RNA. Previous preclinical mouse studies and canine cancer trials used mixed populations of CPMV (containing B, M, and T components), so it is unclear whether the particle types differ in their efficacies. It is known that the CPMV RNA genome contributes to immunostimulation by activation of TLR7. To determine whether the two RNA genomes that have different sizes and unrelated sequences cause different immune stimulation, we compared the therapeutic efficacies of B and M components and unfractionated CPMV in vitro and in mouse cancer models. We found that separated B and M particles behaved similarly to the mixed CPMV, activating innate immune cells to induce the secretion of pro-inflammatory cytokines such as IFNα, IFNγ, IL-6, and IL-12, while inhibiting immunosuppressive cytokines such as TGF-ß and IL-10. In murine models of melanoma and colon cancer, the mixed and separated CPMV particles all significantly reduced tumor growth and prolonged survival with no significant difference. This shows that the specific RNA genomes similarly stimulate the immune system even though B particles have 40% more RNA than M particles; each CPMV particle type can be used as an effective adjuvant against cancer with the same efficacy as native mixed CPMV. From a translational point of view, the use of either B or M component vs the mixed CPMV formulation offers the advantage that separated B or M alone is noninfectious toward plants and thus provides agronomic safety.

Inactivated Cowpea Mosaic Virus for In Situ Vaccination: Differential Efficacy of Formalin vs UV-Inactivated Formulations.

Cowpea mosaic virus (CPMV) has been developed as a promising nanoplatform technology for cancer immunotherapy; when applied as in situ vaccine, CPMV exhibits potent, systemic, and durable efficacy. While CPMV is not infectious to mammals, it is infectious to legumes; therefore, agronomic safety needs to be addressed to broaden the translational application of CPMV. RNA-containing formulations are preferred over RNA-free virus-like particles because the RNA and protein, each, contribute to CPMV's potent antitumor efficacy. We have previously optimized inactivation methods to develop CPMV that contains RNA but is not infectious to plants. We established that inactivated CPMV has reduced efficacy compared to untreated, native CPMV. However, a systematic comparison between native CPMV and different inactivated forms of CPMV was not done. Therefore, in this study, we directly compared the therapeutic efficacies and mechanisms of immune activation of CPMV, ultraviolet- (UV-), and formalin (Form)-inactivated CPMV to explain the differential efficacies. In a B16F10 melanoma mouse tumor model, Form-CPMV suppressed the tumor growth with prolonged survival (there were no statistical differences comparing CPMV and Form-CPMV). In comparison, UV-CPMV inhibited tumor growth significantly but not as well as Form-CPMV or CPMV. The reduced therapeutic efficacy of UV-CPMV is explained by the degree of cross-linking and aggregated state of the RNA, which renders it inaccessible for sensing by Toll-like receptor (TLR) 7/8 to activate immune responses. The mechanistic studies showed that the highly aggregated state of UV-CPMV inhibited TLR7 signaling more so than for the Form-CPMV formulation, reducing the secretion of interleukin-6 (IL-6) and interferon-α (IFN-α), cytokines associated with TLR7 signaling. These findings support the translational development of Form-CPMV as a noninfectious immunotherapeutic agent.

Transcriptomics of Canine Inflammatory Mammary Cancer Treated with Empty Cowpea Mosaic Virus Implicates Neutrophils in Anti-Tumor Immunity.

Canine inflammatory mammary cancer (IMC) is a highly aggressive and lethal cancer in dogs serving as a valuable animal model for its human counterpart, inflammatory breast cancer (IBC), both lacking effective therapies. Intratumoral immunotherapy (IT-IT) with empty cowpea mosaic virus (eCPMV) nanoparticles has shown promising results, demonstrating a reduction in tumor size, longer survival rates, and improved quality of life. This study compares the transcriptomic profiles of tumor samples from female dogs with IMC receiving eCPMV IT-IT and medical therapy (MT) versus MT alone. Transcriptomic analyses, gene expression profiles, signaling pathways, and cell type profiling of immune cell populations in samples from four eCPMV-treated dogs with IMC and four dogs with IMC treated with MT were evaluated using NanoString Technologies using a canine immune-oncology panel. Comparative analyses revealed 34 differentially expressed genes between treated and untreated samples. Five genes (CXCL8, S100A9, CCL20, IL6, and PTGS2) involved in neutrophil recruitment and activation were upregulated in the treated samples, linked to the IL17-signaling pathway. Cell type profiling showed a significant increase in neutrophil populations in the tumor microenvironment after eCPMV treatment. These findings highlight the role of neutrophils in the anti-tumor response mediated by eCPMV IT-IT and suggest eCPMV as a novel therapeutic approach for IBC/IMC.

Cowpea Mosaic Virus Outperforms Other Members of the Secoviridae as In Situ Vaccine for Cancer Immunotherapy.

In situ vaccination for cancer immunotherapy uses intratumoral administration of small molecules, proteins, nanoparticles, or viruses that activate pathogen recognition receptors (PRRs) to reprogram the tumor microenvironment and prime systemic antitumor immunity. Cowpea mosaic virus (CPMV) is a plant virus that─while noninfectious toward mammals─activates mammalian PRRs. Application of CPMV as in situ vaccine (ISV) results in a potent and durable efficacy in tumor mouse models and canine patients; data indicate that CPMV outperforms small molecule PRR agonists and other nonrelated plant viruses and virus-like particles (VLPs). In this work, we set out to compare the potency of CPMV versus other plant viruses from the Secoviridae. We developed protocols to produce and isolate cowpea severe mosaic virus (CPSMV) and tobacco ring spot virus (TRSV) from plants. CPSMV, like CPMV, is a comovirus with genome and protein homology, while TRSV lacks homology and is from the genus nepovirus. When applied as ISV in a mouse model of dermal melanoma (using B16F10 cells and C57Bl6J mice), CPMV outperformed CPSMV and TRSV─again highlighting the unique potency of CPMV. Mechanistically, the increased potency is related to increased signaling through toll-like receptors (TLRs)─in particular, CPMV signals through TLR2, 4, and 7. Using knockout (KO) mouse models, we demonstrate here that all three plant viruses signal through the adaptor molecule MyD88─with CPSMV and TRSV predominantly activating TLR2 and 4. CPMV induced significantly more interferon ß (IFNß) compared to TRSV and CPSMV; therefore, IFNß released upon signaling through TLR7 may be a differentiator for the observed potency of CPMV-ISV. Additionally, CPMV induced a different temporal pattern of intratumoral cytokine generation characterized by significantly increased inflammatory cytokines 4 days after the second of 2 weekly treatments, as if CPMV induced a "memory response". This higher, longer-lasting induction of cytokines may be another key differentiator that explains the unique potency of CPMV-ISV.

Inactivated Cowpea Mosaic Virus in Combination with OX40 Agonist Primes Potent Antitumor Immunity in a Bilateral Melanoma Mouse Model.

Viral immunotherapies are being recognized in cancer treatment, with several currently approved or undergoing clinical testing. While contemporary approaches have focused on oncolytic viral therapies, our efforts center on the development of plant virus-based cancer immunotherapies. In a previous work, we demonstrated the potent efficacy of the cowpea mosaic virus (CPMV), a plant virus that does not replicate in animals, applied as an in situ vaccine. CPMV is an immunostimulatory drug candidate, and intratumoral administration remodels the tumor microenvironment leading to activation of local and systemic antitumor immunity. Efficacy has been demonstrated in multiple tumor mouse models and canine cancer patients. As wild-type CPMV is infectious toward various legumes and because shedding of infectious virus from patients may be an agricultural concern, we developed UV-inactivated CPMV (termed inCPMV) which is not infectious toward plants. We report that as a monotherapy, wild-type CPMV outperforms inCPMV in mouse models of dermal melanoma or disseminated colon cancer. Efficacy of inCPMV is less than that of CPMV and similar to that of RNA-free CPMV. Immunological investigation using knockout mice shows that inCPMV does not signal through TLR7 (toll-like receptor); structure-function studies indicate that the RNA is highly cross-linked and therefore unable to activate TLR7. Wild-type CPMV signals through TLR2, -4, and -7, whereas inCPMV more closely resembles RNA-free CPMV which signals through TLR2 and -4 only. The structural features of inCPMV explain the increased potency of wild-type CPMV through the triple pronged TLR activation. Strikingly, when inCPMV is used in combination with an anti-OX40 agonist antibody (administered systemically), exceptional efficacy was demonstrated in a bilateral B16F10 dermal melanoma model. Combination therapy, with in situ vaccination applied only into the primary tumor, controlled the progression of the secondary, untreated tumors, with 10 out of 14 animals surviving for at least 100 days post tumor challenge without development of recurrence or metastatic disease. This study highlights the potential of inCPMV as an in situ vaccine candidate and demonstrates the power of combined immunotherapy approaches. Strategic immunocombination therapies are the formula for success, and the combination of in situ vaccination strategies along with therapeutic antibodies targeting the cancer immunity cycle is a particularly powerful approach.

Differential effects of CD20+ B cells and PD-L1+ immune cells on pathologic complete response and outcome: comparison between inflammatory breast cancer and locally advanced breast cancer patients.

PURPOSE: This study evaluated epidemiologic and immune factors associated with pathologic complete response (pCR), breast cancer-specific survival (BCSS) and disease-free survival (DFS) outcomes in inflammatory (IBC) and locally advanced breast cancer (LABC) patients. METHODS: Tumor-infiltrating lymphocytes (TILs) and CD20+ B-cell frequencies (CD20+), and PD-L1 expression on tumor (PD-L1+carcinoma cells) and immune (PD-L1+TILs) cells were analyzed by immunohistochemistry along with clinicopathologic factors as modifiers of pCR and outcomes in 221 IBC and 162 LABC patients. Analysis included Kaplan-Meier curves and Cox proportional hazard models. RESULTS: IBC and LABC display similar levels of TILs, CD20+, and combined CD20+ and PD-L1+TILs (CD20+PD-L1+TILs), while LABC contained more PD-L1+TILs and PD-L1+ carcinoma cells. Absence of lymphovascular involvement, high TILs, PD-L1+ carcinoma cells, and combined CD20+ and PD-L1+ carcinoma cells correlated with pCR in IBC and LABC patients. High PD-L1+TILs correlated with pCR only in LABC; less lymph node involvement at diagnosis, CD20+ and CD20+PD-L1+TILs correlated with pCR only in IBC (P < 0.04, all comparisons). Achievement of pCR in IBC and LABC patients correlated with BCSS and DFS (P < 0.02). In multivariate analyses, pCR remained an independent prognostic factor of improved DFS in IBC and LABC patients, but of BCSS in only LABC. CD20+PD-L1+TILs remained an independent prognostic factor of improved DFS and BCSS only in IBC. CONCLUSION: CD20+PD-L1+TILs are an independent prognostic biomarker of improved outcomes in IBC, but not LABC. Selecting IBC patients by CD20 and PD-L1 status could stratify patients and potentially identify those in whom activating CD20 agents and anti-PD-1/PD-L1 therapy could be explored.

Endosomal toll-like receptors play a key role in activation of primary human monocytes by cowpea mosaic virus.

The plant virus, cowpea mosaic virus (CPMV), has demonstrated a remarkable capacity to induce anti-tumour immune responses following direct administration into solid tumours. The molecular pathways that account for these effects and the capacity of CPMV to activate human cells are not well defined. Here, we examine the ability of CPMV particles to activate human monocytes, dendritic cells (DCs) and macrophages. Monocytes in peripheral blood mononuclear cell cultures and purified CD14+ monocytes were readily activated by CPMV in vitro, leading to induction of HLA-DR, CD86, PD-L1, IL-15R and CXCL10 expression. Monocytes released chemokines, CXCL10, MIP-1α and MIP-1ß into cell culture supernatants after incubation with CPMV. DC subsets (pDC and mDC) and monocyte-derived macrophages also demonstrated evidence of activation after incubation with CPMV. Inhibitors of spleen tyrosine kinase (SYK), endocytosis or endocytic acidification impaired the capacity of CPMV to activate monocytes. Furthermore, CPMV activation of monocytes was partially blocked by a TLR7/8 antagonist. These data demonstrate that CPMV activates human monocytes in a manner dependent on SYK signalling, endosomal acidification and with an important contribution from TLR7/8 recognition.

Using nanoparticles for in situ vaccination against cancer: mechanisms and immunotherapy benefits.

Immunotherapy to treat cancer is now an established clinical approach. Immunotherapy can be applied systemically, as done with checkpoint blockade antibodies, but it can also be injected directly into identified tumors, in a strategy of in situ vaccination (ISV). ISV is designed to stimulate a strong local antitumor immune response involving both innate and adaptive immune cells, and through this generate a systemic antitumor immune response against metastatic tumors. A variety of ISVs have been utilized to generate an immunostimulatory tumor microenvironment (TME). These include attenuated microorganisms, recombinant proteins, small molecules, physical disruptors of TME (alternating magnetic and focused ultrasound heating, photothermal therapy, and radiotherapy), and more recently nanoparticles (NPs). NPs are attractive and unique since they can load multiple drugs or other reagents to influence immune and cancer cell functions in the TME, affording a unique opportunity to stimulate antitumor immunity. Here, we describe the NP-ISV therapeutic mechanisms, review chemically synthesized NPs (i.e., liposomes, polymeric, chitosan-based, inorganic NPs, etc.), biologically derived NPs (virus and bacteria-based NPs), and energy-activated NP-ISVs in the context of their use as local ISV. Data suggests that NP-ISVs can enhance outcomes of immunotherapeutic regimens including those utilizing tumor hyperthermia and checkpoint blockade therapies.

In situ vaccination with nanoparticles for cancer immunotherapy: understanding the immunology.

FDA approval of anti-CTLA4 in 2011 for melanoma immunotherapy was paradigm shifting and dramatically accelerated cancer immunotherapy research. The investment and effort have been exceptionally large, with a commensurate impressive pace of discovery. Historical and current research has validated the following key points: tumors are recognized by the immune system; tumors develop an immunosuppressive environment which suppresses the antitumor immune response; successful immunotherapy must overcome that tumor-mediated immunosuppression. While cancer immunotherapy research expanded, a parallel effort developing nanoparticles (NP) for cancer diagnosis and therapy also received major investment and expanded. Initially the two efforts appeared to have minimal synergy. Systemically administered nanoparticles are rapidly ingested by phagocytic leukocytes, and therefore nanotechnologists developed strategies to avoid NP ingestion by leukocytes in order to accomplish nanoparticle accumulation in tumors rather than liver and spleen. Recently, nanotechnology and cancer immunotherapy have increasingly merged since phagocytic leukocytes are the key to reversing the local tumor immunosuppression and the tendency of NP to be phagocytosed can be exploited to manipulate phagocytes for immunotherapy. This review focuses on in situ vaccination (ISV), an immunotherapy approach that can utilize direct injection of immunostimulatory reagents, including NPs, into tumors to disrupt the local immunosuppression, stimulate effective immune response against the treated tumor, and most importantly, generate a systemic antitumor immune response to eliminate metastatic tumors. While there are many specific options for using NP for ISV (reviewed further in this special issue), this review focuses on immunology concepts needed to understand and design successful NP ISV approaches.

In-situ vaccination using focused ultrasound heating and anti-CD-40 agonistic antibody enhances T-cell mediated local and abscopal effects in murine melanoma.

The success of melanoma immunotherapy is dependent on the presence of activated and functional T-cells in tumors. The objective of this study was to investigate the impact of local-focused ultrasound (FUS) heating (∼42-45 °C) and in-situ anti-CD-40 agonistic antibody in enhancing T-cell function for melanoma immunotherapy. We compared the following groups of mice with bilateral flank B16 F10 melanoma: (1) Control, (2) FUS, (3) CD-40, and (4) CD-40 + FUS (FUS40). FUS heating was applied for ∼15 min in right flank tumor, and intratumoral injections of CD-40 were performed sequentially within 4 h. A total of 3 FUS and 4 anti-CD-40 treatments were administered unilaterally 3 days apart. Mice were sacrificed 30 days post-inoculation, and the treated tumor and spleen tissues were profiled for T-cell function and macrophage polarization. Compared to all other groups, histology and flow cytometry showed that FUS40 increased the population of tumor-specific CD-4+ and CD-8+ T cells rich in Granzyme B+, interleukin-2 (IL-2) and IFN-γ production and poor in PD-1 expression. In addition, FUS40 promoted the infiltration of tumor-suppressing M1 phenotype macrophages in the treated mice. The resultant immune-enhancing effects of FUS40 suppressed B16 melanoma growth at the treated site by 2-3-folds compared to control, FUS, and CD-40, and also achieved significant abscopal effects in untreated tumors relative to CD40 alone. Additionally, the local FUS40 prevented adverse liver toxicities in the treated mice. Our study suggests that combined FUS and CD-40 can enhance T-cell and macrophage functions to aid effective melanoma immunotherapy.

Immunogenetic effects of low dose (CEM43 30) magnetic nanoparticle hyperthermia and radiation in melanoma cells.

Objective: In this in vitro study we have used an RNA quantification technique, nanoString, and a conventional protein analysis technique (Western Blot) to assess the genetic and protein expression of B16 murine melanoma cells following a modest magnetic nanoparticle hyperthermia (mNPH) dose equivalent to 30 minutes @ 43°C (CEM43 30) and/or a clinically relevant 8 Gy radiation dose.Methods: Melanoma cells with mNPs(2.5 µg Fe/106 cells) were pelleted and exposed to an alternating magnetic field (AMF) to generate the targeted thermal dose. Thermal dose was accurately monitored by a fiber optic probe and automatically maintained at CEM43 30. All cells were harvested 24 hours after treatment.Results: The mNPH dose demonstrated notable elevations in the thermotolerance/immunogenic HSP70 gene and a number of chemoattractant and toll-like receptor gene pathways. The 8 Gy dose also upregulated a number of important immune and cytotoxic genetic and protein pathways. However, the mNPH/radiation combination was the most effective stimulator of a wide variety of immune and cytotoxic genes including HSP70, cancer regulating chemokines CXCL10, CXCL11, the T-cell trafficking chemokine CXCR3, innate immune activators TLR3, TLR4, the MDM2 and mTOR negative regulator of p53, the pro-apoptotic protein PUMA, and the cell death receptor Fas. Importantly a number of the genetic changes were accurately validated by protein expression changes, i.e., HSP70, p-mTOR, p-MDM2.Conclusion: These results not only show that low dose mNPH and radiation independently increase the expression of important immune and cytotoxic genes but that the effect is greatly enhanced when they are used in combination.

Beta-glucan-induced inflammatory monocytes mediate antitumor efficacy in the murine lung.

ß-Glucan is a naturally occurring glucose polysaccharide with immunostimulatory activity in both infection and malignancy. ß-Glucan's antitumor effects have been attributed to the enhancement of complement receptor 3-dependent cellular cytotoxicity, as well as modulation of suppressive and stimulatory myeloid subsets, which in turn enhances antitumor T cell immunity. In the present study, we demonstrate antitumor efficacy of yeast-derived ß-glucan particles (YGP) in a model of metastatic-like melanoma in the lung, through a mechanism that is independent of previously reported ß-glucan-mediated antitumor pathways. Notably, efficacy is independent of adaptive immunity, but requires inflammatory monocytes. YGP-activated monocytes mediated direct cytotoxicity against tumor cells in vitro, and systemic YGP treatment upregulated inflammatory mediators, including TNFα, M-CSF, and CCL2, in the lungs. Collectively, these studies identify a novel role for inflammatory monocytes in ß-glucan-mediated antitumor efficacy, and expand the understanding of how this immunomodulator can be used to generate beneficial immune responses against metastatic disease.

In Situ Vaccination with Cowpea vs Tobacco Mosaic Virus against Melanoma.

Cancer immunotherapy approaches have emerged as novel treatment regimens against cancer. A particularly interesting avenue is the concept of in situ vaccination, where immunostimulatory agents are introduced into an identified tumor to overcome local immunosuppression and, if successful, mount systemic antitumor immunity. We had previously shown that nanoparticles from cowpea mosaic virus (CPMV) are highly potent in inducing long-lasting antitumor immunity when used as an in situ vaccine in various tumor mouse models. Here we asked whether the nanoparticles from tobacco mosaic virus (TMV) could also be applied as an in situ vaccine and, if so, whether efficacy or mechanism of immune-activation would be affected by the nanoparticle size (300 × 18 nm native TMV vs 50 × 18 nm short TMV nanorods), shape (nanorods vs spherical TMV, termed SNP), or state of assembly (assembled TMV rod vs free coat protein, CP). Our studies indicate that CPMV, but less so TMV, elicits potent antitumor immunity after intratumoral treatment of dermal melanoma (B16F10 using C57BL/6 mice). TMV and TMVshort slowed tumor growth and increased survival time, however, at significantly lower potency compared to that of CPMV. There were no apparent differences between TMV, TMVshort, or the SNP indicating that the aspect ratio does not necessarily play a role in plant viral in situ vaccines. The free CPs did not elicit an antitumor response or immunostimulation, which may indicate that a multivalent assembly is required to trigger an innate immune recognition and activation. Differential potency of CPMV vs TMV can be explained with differences in immune-activation: data indicate that CPMV stimulates an antitumor response through recruitment of monocytes into the tumor microenvironment (TME), establishing signaling through the IFN-γ pathway, which also leads to recruitment of tumor-infiltrated neutrophils (TINs) and natural killer (NK) cells. Furthermore, the priming of the innate immune system also mounts an adaptive response with CD4+ and CD8+ T cell recruitment and establishment of effector memory cells. While the TMV treatment also lead to the recruitment of innate immune cells as well as T cells (although to a lesser degree), key differences were noted in cyto/chemokine profiling with TMV inducing a potent immune response early on characterized by strong pro-inflammatory cytokines, primarily IL-6. Together, data indicate that some plant viral nanotechnology platforms are more suitable for application as in situ vaccines than others; understanding the intricate differences and underlying mechanism of immune-activation may set the stage for clinical development of these technologies.

Treatment of Canine Oral Melanoma with Nanotechnology-Based Immunotherapy and Radiation.

The presence and benefit of a radiation therapy-associated immune reaction is of great interest as the overall interest in cancer immunotherapy expands. The pathological assessment of irradiated tumors rarely demonstrates consistent immune or inflammatory response. More recent information, primarily associated with the "abscopal effect", suggests a subtle radiation-based systemic immune response may be more common and have more therapeutic potential than previously believed. However, to be of consistent value, the immune stimulatory potential of radiation therapy (RT) will clearly need to be supported by combination with other immunotherapy efforts. In this study, using a spontaneous canine oral melanoma model, we have assessed the efficacy and tumor immunopathology of two nanotechnology-based immune adjuvants combined with RT. The immune adjuvants were administered intratumorally, in an approach termed "in situ vaccination", that puts immunostimulatory reagents into a recognized tumor and utilizes the endogenous antigens in the tumor as the antigens in the antigen/adjuvant combination that constitutes a vaccine. The radiation treatment consisted of a local 6 × 6 Gy tumor regimen given over a 12 day period. The immune adjuvants were a plant-based virus-like nanoparticle (VLP) and a 110 nm diameter magnetic iron oxide nanoparticle (mNPH) that was activated with an alternating magnetic field (AMF) to produce moderate heat (43 °C/60 min). The RT was used alone or combined with one or both adjuvants. The VLP (4 × 200 µg) and mNPH (2 × 7.5 mg/gram tumor) were delivered intratumorally respectively during the RT regimen. All patients received a diagnostic biopsy and CT-based 3-D radiation treatment plan prior to initiating therapy. Patients were assessed clinically 14-21 days post-treatment, monthly for 3 months following treatment, and bimonthly, thereafter. Immunohistopathologic assessment of the tumors was performed before and 14-21 days following treatment. Results suggest that addition of VLPs and/or mNPH to a hypofractionated radiation regimen increases the immune cell infiltration in the tumor, extends the tumor control interval, and has important systemic therapeutic potential.

Combination of Plant Virus Nanoparticle-Based in Situ Vaccination with Chemotherapy Potentiates Antitumor Response.

Immunotherapeutics are gaining more traction in the armamentarium used to combat cancer. Specifically, in situ vaccination strategies have gained interest because of their ability to alter the tumor microenvironment to an antitumor state. Herein, we investigate whether flexuous plant virus-based nanoparticles formed by the potato virus X (PVX) can be used as an immunotherapeutic for in situ vaccine monotherapy. We further developed dual chemo-immunotherapeutics by incorporating doxorubicin (DOX) into PVX yielding a dual-functional nanoparticle (PVX-DOX) or by coadministration of the two therapeutic regimes, PVX immunotherapy and DOX chemotherapy (PVX+DOX). In the context of B16F10 melanoma, PVX was able to elicit delayed tumor progression when administered as an intratumoral in situ vaccine. Furthermore, the coadministration of DOX via PVX+DOX enhanced the response of the PVX monotherapy through increased survival, which was also represented in the enhanced antitumor cytokine/chemokine profile stimulated by PVX+DOX when compared to PVX or DOX alone. Importantly, coadministered PVX+DOX was better for in situ vaccination than PVX loaded with DOX (PVX-DOX). Whereas the nanomedicine field strives to design multifunctional nanoparticles that integrate several functions and therapeutic regimens into a single nanoparticle, our data suggest a paradigm shift; some therapeutics may need to be administered separately to synergize and achieve the most potent therapeutic outcome. Altogether, our studies show that development of plant viral nanoparticles for in situ vaccines for treatment is a possibility, and dual mechanistic therapeutics can increase efficacy. Nonetheless, combining immunotherapeutics with cytolytic chemotherapy requires detailed investigation to inform optimal integration of cytolytic and immunotherapies and maximize synergy and efficacy.

Myeloid Acyl-CoA:Cholesterol Acyltransferase 1 Deficiency Reduces Lesion Macrophage Content and Suppresses Atherosclerosis Progression.

Acyl-CoA:cholesterol acyltransferase 1 (Acat1) converts cellular cholesterol to cholesteryl esters and is considered a drug target for treating atherosclerosis. However, in mouse models for atherosclerosis, global Acat1 knockout (Acat1(-/-)) did not prevent lesion development. Acat1(-/-) increased apoptosis within lesions and led to several additional undesirable phenotypes, including hair loss, dry eye, leukocytosis, xanthomatosis, and a reduced life span. To determine the roles of Acat1 in monocytes/macrophages in atherosclerosis, we produced a myeloid-specific Acat1 knockout (Acat1(-M/-M)) mouse and showed that, in the Apoe knockout (Apoe(-/-)) mouse model for atherosclerosis, Acat1(-M/-M) decreased the plaque area and reduced lesion size without causing leukocytosis, dry eye, hair loss, or a reduced life span. Acat1(-M/-M) enhanced xanthomatosis in apoe(-/-) mice, a skin disease that is not associated with diet-induced atherosclerosis in humans. Analyses of atherosclerotic lesions showed that Acat1(-M/-M) reduced macrophage numbers and diminished the cholesterol and cholesteryl ester load without causing detectable apoptotic cell death. Leukocyte migration analysis in vivo showed that Acat1(-M/-M) caused much fewer leukocytes to appear at the activated endothelium. Studies in inflammatory (Ly6C(hi)-positive) monocytes and in cultured macrophages showed that inhibiting ACAT1 by gene knockout or by pharmacological inhibition caused a significant decrease in integrin ß 1 (CD29) expression in activated monocytes/macrophages. The sparse presence of lesion macrophages without Acat1 can therefore, in part, be attributed to decreased interaction between inflammatory monocytes/macrophages lacking Acat1 and the activated endothelium. We conclude that targeting ACAT1 in a myeloid cell lineage suppresses atherosclerosis progression while avoiding many of the undesirable side effects caused by global Acat1 inhibition.

Adaptive immunity programmes in breast cancer.

The role of the immune system in shaping cancer development and patient prognosis has recently become an area of intense focus in industry and academia. Harnessing the adaptive arm of the immune system for tumour eradication has shown great promise in a variety of tumour types. Differences between tissues, however, necessitate a greater understanding of the adaptive immunity programmes that are active within each tumour type. In breast cancer, adaptive immune programmes play diverse roles depending on the cellular infiltration found in each tumour. Cytotoxic T lymphocytes and T helper type 1 cells can induce tumour eradication, whereas regulatory T cells and T helper type 2 cells are known to be involved in tumour-promoting immunosuppressive responses. Complicating these matters, heterogeneous expression of hormone receptors and growth factors in different tumours leads to disparate, patient-specific adaptive immune responses. Despite this non-conformity in adaptive immune behaviours, encouraging basic and clinical results have been observed that suggest a role for immunotherapeutic approaches in breast cancer. Here, we review the literature pertaining to the adaptive immune response in breast cancer, summarize the primary findings relating to the breast tumour's biology, and discuss potential clinical immunotherapies.