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Muscle fiber cross-sectional area (CSA) and proportion of different fiber types are important determinants of muscle function and overall metabolism. Genetic variation plays a substantial role in phenotypic variation of these traits; however, the underlying genes remain poorly understood. This study aimed to map quantitative trait loci (QTL) affecting differences in soleus muscle fiber traits between the LG/J and SM/J mouse strains. Fiber number, CSA, and proportion of oxidative type I fibers were assessed in the soleus of 334 genotyped female and male mice of the F34 generation of advanced intercross lines (AIL) derived from the LG/J and SM/J strains. To increase the QTL detection power, these data were combined with 94 soleus samples from the F2 intercross of the same strains. Transcriptome of the soleus muscle of LG/J and SM/J females was analyzed by microarray. Genome-wide association analysis mapped four QTL (genome-wide P < 0.05) affecting the properties of muscle fibers to chromosome 2, 3, 4, and 11. A 1.5-LOD QTL support interval ranged between 2.36 and 4.67 Mb. On the basis of the genomic sequence information and functional and transcriptome data, we identified candidate genes for each of these QTL. The combination of analyses in F2 and F34 AIL populations with transcriptome and genomic sequence data in the parental strains is an effective strategy for refining QTL and nomination of the candidate genes.

Christian Ndong,1 Seiko Toraya-Brown,2 Katsiaryna Kekalo,1 Ian Baker,1 Tillman U Gerngross,1,3,4 Steven N Fiering,2,5,6 Karl E Griswold1,3,6 1Thayer School of Engineering, Dartmouth, Hanover, NH, USA; 2Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA; 3Department of Biological Sciences, Dartmouth, Hanover, NH, USA; 4Department of Chemistry, Dartmouth, Hanover, NH, USA; 5Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, NH, USA; 6Norris Cotton Cancer Center, Lebanon, NH, USA Abstract: Active molecular targeting has become an important aspect of nanoparticle development for oncology indications. Here, we describe molecular targeting of iron oxide nanoparticles (IONPs) to the folate receptor alpha (FOLRα) using an engineered antibody fragment (Ffab). Compared to control nanoparticles targeting the non-relevant botulinum toxin, the Ffab-IONP constructs selectively accumulated on FOLRα-overexpressing cancer cells in vitro, where they exhibited the capacity to internalize into intracellular vesicles. Similarly, Ffab-IONPs homed to FOLRα-positive tumors upon intraperitoneal administration in an orthotopic murine xenograft model of ovarian cancer, whereas negative control particles showed no detectable tumor accumulation. Interestingly, Ffab-IONPs built with custom 120 nm nanoparticles exhibited lower in vitro targeting efficiency when compared to those built with commercially sourced 180 nm nanoparticles. In vivo, however, the two Ffab-IONP platforms achieved equivalent tumor homing, although the smaller 120 nm IONPs were more prone to liver sequestration. Overall, the results show that Ffab-mediated targeting of IONPs yields specific, high-level accumulation within cancer cells, and this fact suggests that Ffab-IONPs could have future utility in ovarian cancer diagnostics and therapy. Keywords: nanoparticle targeting, antibody fragment, biodistribution, ovarian cancer

The phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates many important cellular functions. The functional impact of deregulating the PIK3CA gene, encoding the p110α catalytic subunit of PI3K, is validated by frequent gain of function mutations in a range of human cancers. We generated a mouse model with an inducible constitutively active form of PI3K. In this model Cre recombinase activates expression of a myristoylated form of p110α (myr-p110α). The myristoylated version of p110α brings the protein to the cytoplasmic side of the cell membrane, which mimics the normal activation mechanism for the p110α catalytic subunit and activates the PI3K enzyme. Constitutively activated PI3K signaling induced by myr-p110α in all cells of the developing mouse caused lethality during embryonic development. Transgenic Cre;myr-p110α heterozygous embryos displayed morphological malformation and poor vascular development with extremely dilated blood vessels and hemorrhage in the embryo and the extraembryonic yolk sac. Previous studies demonstrated that loss of p110α during embryonic development causes angiogenic disruption and here we show that constitutive activation of p110α by gain of function mutation during development also disrupts vasculogenesis/angiogenesis in what appears to be a similar manner. These finding demonstrate the importance of tight regulation of PI3K signaling during embryonic vasculogenesis/angiogenesis.

Neoadjuvant intratumoral (IT) therapy could amplify the weak responses to checkpoint blockade therapy observed in breast cancer (BC). In this study, we administered neoadjuvant IT anti-canine PD-1 therapy (IT acPD-1) alone or combined with IT cowpea mosaic virus therapy (IT CPMV/acPD-1) to companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. CMC patients treated weekly with acPD-1 (n = 3) or CPMV/acPD-1 (n = 3) for four weeks or with CPMV/acPD-1 (n = 3 patients not candidates for surgery) for up to 11 weeks did not experience immune-related adverse events. We found that acPD-1 and CPMV/acPD-1 injections resulted in tumor control and a reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of treated dogs. In two metastatic CMC patients, CPMV/acPD-1 treatments resulted in the control and reduction of established lung metastases. CPMV/acPD-1 treatments were associated with altered gene expression related to TLR1-4 signaling and complement pathways. These novel therapies could be effective for CMC patients. Owing to the extensive similarities between CMC and human BC, IT CPMV combined with approved anti-PD-1 therapies could be a novel and effective immunotherapy to treat local BC and suppress metastatic BC.

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 ( Katnal2Δ 17 ) 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 Katnal2 Δ17 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 Katnal2 Δ 17 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., Competing Interests: Competing interests statement:The authors declare no competing interest.

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., (© 2024. The Author(s).)

Implantable polymeric hydrogels loaded with immunostimulatory cowpea mosaic virus (CPMV) were fabricated using digital light processing (DLP) printing technology. The CPMV-laden hydrogels were surgically implanted into the peritoneal cavity to serve as depots for cancer slow-release immunotherapy. Sustained release of CPMV within the intraperitoneal space alleviates the need for repeated dosing and we demonstrated efficacy against ovarian cancer in a metastatic mouse model., Competing Interests: The authors declare the following competing financial interest(s): Dr Steinmetz and Dr Fiering are co-founders and have equity and financial interest in Mosaic ImmunoEnginering Inc. Dr Steinmetz is a co-founder of, and serves as manager of Pokometz Scientific LLC, under which she is a paid consultant to Mosaic ImmunoEngineering Inc., Flagship Labs 95 Inc., and Arana Biosciences Inc. The other authors declare no potential COI., (This journal is © The Royal Society of Chemistry.)

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.

The lack of optimal models to evaluate novel agents is delaying the development of effective immunotherapies against human breast cancer (BC). In this prospective open label study, we applied neoadjuvant intratumoral immunotherapy with empty cowpea mosaic virus-like particles (eCPMV) to 11 companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. We found that two neoadjuvant intratumoral eCPMV injections resulted in tumor reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of injected dogs. Tumor reduction was independent of clinical stage, tumor size, histopathologic grade, and tumor molecular subtype. RNA-seq-based analysis of injected tumors indicated a decrease in DNA replication activity and an increase in activated dendritic cell infiltration in the tumor microenvironment. Immunohistochemistry analysis demonstrated significant intratumoral increases in neutrophils, T and B lymphocytes, and plasma cells. eCPMV intratumoral immunotherapy demonstrated antitumor efficacy without any adverse effects. This novel immunotherapy has the potential for improving outcomes for human BC patients.

Energy-based focal therapy (FT) uses targeted, minimally invasive procedures to destroy tumors while preserving normal tissue and function. There is strong emerging interest in understanding how systemic immunity against the tumor can occur with cancer immunotherapy, most notably immune checkpoint inhibitors (ICI). The motivation for combining FT and ICI in cancer management relies on the synergy between the two different therapies: FT complements ICI by reducing tumor burden, increasing objective response rate, and reducing side effects of ICI; ICI supplements FT by reducing local recurrence, controlling distal metastases, and providing long-term protection. This combinatorial strategy has shown promising results in preclinical study (since 2004) and the clinical trials (since 2011). Understanding the synergy calls for understanding the physics and biology behind the two different therapies with distinctive mechanisms of action. In this review, we introduce different types of energy-based FT by covering the biophysics of tissue-energy interaction and present the immunomodulatory properties of FT. We discuss the basis of cancer immunotherapy with the emphasis on ICI. We examine the approaches researchers have been using and the results from both preclinical models and clinical trials from our exhaustive literature research. Finally, the challenges of the combinatory strategy and opportunities of future research is discussed extensively., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jiang, Fiering and Shao.)

Immunization approaches are designed to stimulate the immune system and eliminate the tumor. Studies indicate that cancer immunization combined with certain chemotherapeutics and immunostimulatory agents can improve outcomes. Chemotherapeutics-based immunogenic cell death makes the tumor more recognizable by the immune system. In situ vaccination (ISV) utilizes established tumors as antigen sources and directly applies an immune adjuvant to the tumor to reverse a cold tumor microenvironment to a hot one. Immunogenic cell death and ISV highlight for the immune system the tumor antigens that are recognizable by immune cells and support a T-cell attack of the tumor cells. This review presents the concept of immunogenic apoptosis and ISV as a powerful platform for cancer immunization.

The tumor microenvironment (TME) within and around a tumor is a complex interacting mixture of tumor cells with various stromal cells, including endothelial cells, fibroblasts, and immune cells. In the early steps of tumor formation, the local microenvironment tends to oppose carcinogenesis, while with cancer progression, the microenvironment skews into a protumoral TME and the tumor influences stromal cells to provide tumor-supporting functions. The creation and development of cancer are dependent on escape from immune recognition predominantly by influencing stromal cells, particularly immune cells, to suppress antitumor immunity. This overall process is generally called immunoediting and has been categorized into three phases; elimination, equilibrium, and escape. Interaction of tumor cells with stromal cells in the TME is mediated generally by cell-to-cell contact, cytokines, growth factors, and extracellular vesicles (EVs). The least well studied are EVs (especially exosomes), which are nanoparticle-sized bilayer membrane vesicles released by many cell types that participate in cell/cell communication. EVs carry various proteins, nucleic acids, lipids, and small molecules that influence cells that ingest the EVs. Tumor-derived extracellular vesicles (TEVs) play a significant role in every stage of immunoediting, and their cargoes change from immune-activating in the early stages of immunoediting into immunosuppressing in the escape phase. In addition, their cargos change with different treatments or stress conditions and can be influenced to be more immune stimulatory against cancer. This review focuses on the emerging understanding of how TEVs affect the differentiation and effector functions of stromal cells and their role in immunoediting, from the early stages of immunoediting to immune escape. Consideration of how TEVs can be therapeutically utilized includes different treatments that can modify TEV to support cancer immunotherapy.

Background: In situ vaccination (ISV) is a cancer immunotherapy strategy in which immunostimulatory reagents are introduced directly into a tumor to stimulate antitumor immunity both against the treated tumor and systemically against untreated tumors. Recently, we showed that cowpea mosaic virus (CPMV) is a potent multi-toll-like receptor (TLR) agonist with potent efficacy for treating tumors in mice and dogs by ISV. However, ISV with CPMV alone does not uniformly treat all mouse tumor models tested, however this can be overcome through strategic combinations. More insight is needed to delineate potency and mechanism of systemic antitumor immunity and abscopal effect., Method: We investigated the systemic efficacy (abscopal effect) of CPMV ISV with a two-tumor mouse model using murine tumor lines B16F10, 4T1, CT26 and MC38. Flow cytometry identified changes in cell populations responsible for systemic efficacy of CPMV. Transgenic knockout mice and depleting antibodies validated the role of relevant candidate cell populations and cytokines. We evaluated these findings and engineered a multicomponent combination therapy to specifically target the candidate cell population and investigated its systemic efficacy, acquired resistance and immunological memory in mouse models., Results: ISV with CPMV induces systemic antitumor T-cell-mediated immunity that inhibits growth of untreated tumors and requires conventional type-1 dendritic cells (cDC1s). Furthermore, using multiple tumor mouse models resistant to anti-programmed death 1 (PD-1) therapy, we tested the hypothesis that CPMV along with local activation of antigen-presenting cells with agonistic anti-CD40 can synergize and strengthen antitumor efficacy. Indeed, this combination ISV strategy induces an influx of CD8 + T cells, triggers regression in both treated local and untreated distant tumors and potentiates tumor responses to anti-PD-1 therapy. Moreover, serial ISV overcomes resistance to anti-PD-1 therapy and establishes tumor-specific immunological memory., Conclusions: These findings provide new insights into in situ TLR activation and cDC1 recruitment as effective strategies to overcome resistance to immunotherapy in treated and untreated tumors., Competing Interests: Competing interests: SF and NFS are co-founders of and have a financial relationship with Mosaic Immunoengineering, which may be considered potential competing interest., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Inflammatory breast cancer (IBC) is highly metastatic at the onset of the disease with no IBC-specific treatments, resulting in dismal patient survival. IBC treatment is a clear unmet clinical need. This commentary highlights findings from a recent seminal approach in which pembrolizumab, a checkpoint inhibitor against programmed cell death protein 1 (PD-1), was provided to a triple-negative IBC patient as a neoadjuvant immune therapy combined with anthracycline-taxane-based chemotherapy. We highlight the findings of the case report and offer a perspective on taking a proactive approach to deploy approved immune checkpoint inhibitors. On the basis of our recently published research study, we propose in situ vaccination with direct injection of immunostimulatory agents into the tumor as an option to improve outcomes safely, effectively, and economically for IBC patients.

Background: Inflammatory mammary cancer (IMC), the counterpart of human inflammatory breast cancer (IBC), is the deadliest form of canine mammary tumors. IMC patients lack specific therapy and have poor outcomes. This proof-of-principle preclinical study evaluated the efficacy, safety, and effect on survival of neoadjuvant intratumoral (in situ) empty cowpea mosaic virus (eCPMV) immunotherapy in companion dogs diagnosed with IMC., Methods: Ten IMC-bearing dogs were enrolled in the study. Five dogs received medical therapy, and five received weekly neoadjuvant in situ eCPMV immunotherapy (0.2-0.4 mg per injection) and medical therapy after the second eCPMV injection. Efficacy was evaluated by reduction of tumor growth; safety by hematological and biochemistry changes in blood and plasma; and patient outcome by survival analysis. eCPMV-induced immune changes in blood cells were analyzed by flow cytometry; changes in the tumor microenvironment were evaluated by CD3 (T lymphocytes), CD20 (B lymphocytes), FoxP3 (Treg lymphocytes), myeloperoxidase (MPO; neutrophils), Ki-67 (proliferation index, PI; tumor cell proliferation), and Cleaved Caspase-3 (CC-3; apoptosis) immunohistochemistry., Results: Two neoadjuvant in situ eCPMV injections resulted in tumor shrinkage in all patients by day 14 without systemic adverse events. Although surgery for IMC is generally not an option, reduction in tumor size allowed surgery in two IMC patients. In peripheral blood, in situ eCPMV immunotherapy was associated with a significant decrease of Treg + /CD8 + ratio and changes in CD8 + Granzyme B + T cells, which behave as a lagging predictive biomarker. In the TME, higher neutrophilic infiltration and MPO expression, lower tumor Ki-67 PI, increase in CD3 + lymphocytes, decrease in FoxP3 + /CD3 + ratio (p < 0.04 for all comparisons), and no changes in CC-3 + immunostainings were observed in post-treatment tumor tissues when compared with pretreatment tumor samples. eCPMV-treated IMC patients had a statistically significant (p=0.033) improved overall survival than patients treated with medical therapy., Conclusions: Neoadjuvant in situ eCPMV immunotherapy demonstrated anti-tumor efficacy and improved survival in IMC patients without systemic adverse effects. eCPMV-induced changes in immune cells point to neutrophils as a driver of immune response. Neoadjuvant in situ eCPMV immunotherapy could be a groundbreaking immunotherapy for canine IMC and a potential future immunotherapy for human IBC patients., Competing Interests: Competing interests: NFS and SF are co-founders of, have equity in, and have a financial interest with Mosaic ImmunoEngineering Inc. SF serves as scientific advisor and paid consultant to Mosaic; NFS serves as Director, Board Member, and Acting Chief Scientific Officer, and paid consultant to Mosaic. JvB is a cofounder and has financial interest in InCephalo AG and is a part-time employee at InCephalo. The other authors declare no potential conflicts of interest., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Cancer immunotherapies using plant virus nanoparticles (PVNPs) have achieved considerable success in preclinical studies. PVNP based nanoplatforms can be endogenous immune adjuvants and act as nanocarriers that stabilize and deliver cancer antigens and exogenous immune adjuvants. Although they do not infect mammalian cells, PVNPs are viruses and they are variably recognized by pathogen pattern recognition receptors (PRR), activate innate immune cells including antigen-presenting cells (APCs), and increase the expression of costimulatory molecules. Novel immunotherapy strategies use them as in situ vaccines (ISV) that can effectively inhibit tumor growth after intratumoral administration and generate expanded systemic antitumor immunity. PVNPs combined with other tumor immunotherapeutic options and other modalities of oncotherapy can improve both local and systemic anti-tumor immune responses. While not yet in clinical trials in humans, there is accelerating interest and research of the potential of PVNPs for ISV immune therapy for cancer. Thus, antitumor efficacy of PVNPs by themselves, or loaded with soluble toll-like receptor (TLR) agonists and/or cancer antigens, will likely enter human trials over the next few years and potentially contribute to next-generation antitumor immune-based therapies., Competing Interests: Conflict of Interest Dr. Fiering is cofounder and has a financial interest in Mosaic Immunoengineering Inc.

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cowpea mosaic virus (CPMV), a non-enveloped plant virus, and empty CPMV (eCPMV), a virus-like particle (VLP) composed of CPMV capsid without nucleic acids, are potent in situ cancer vaccines when administered intratumorally (I.T.). However, it is unclear how immune cells recognize these nanoparticles and why they are immunogenic, which was investigated in this study. CPMV generated stronger selective induction of cytokines and chemokines in naïve mouse splenocytes and exhibited more potent anti-tumor efficacy than eCPMV. MyD88 is required for both CPMV- and eCPMV-elicited immune responses. Screening with human embryonic kidney (HEK)-293 cell toll-like receptor (TLR) reporter assays along with experiments in corresponding TLR-/- mice indicated CPMV and eCPMV capsids are recognized by MyD88-dependent TLR2 and TLR4. CPMV, but not eCPMV, is additionally recognized by TLR7. Secretion of type I interferons (IFNs), which requires the interaction between TLR7 and encapsulated single-stranded RNAs (ssRNAs), is critical to CPMV's better efficacy. The same recognition mechanisms are also functional in human peripheral blood mononuclear cells (PBMCs). Overall, these findings link CPMV immunotherapy efficacy with molecular recognition, provide rationale for how to develop more potent viral particles, accentuate the value of multi-TLR agonists as in situ cancer vaccines, and highlight the functional importance of type I IFNs for in situ vaccination., (Copyright © 2021. Published by Elsevier Ltd.)