Search

Heterogeneity of the tumor microenvironment (TME) and the lack of a definite targetable receptor in triple-negative breast cancer (TNBC) has carved a niche for this cancer as a particularly therapeutically challenging form of breast cancer. However, recent advances in high-throughput genomic analysis have provided new insights into the unique microenvironment and defining characteristics of various subsets of TNBC. This improved understanding has contributed to the development of novel therapeutic strategies including targeted therapies such as PARP inhibitors and CDK inhibitors. Moreover, the recent FDA approval of the immune checkpoint inhibitor against programmed cell death protein 1 (PD-1), pembrolizumab and atezolizumab, holds the promise of improving the quality of life and increasing the overall survival of TNBC patients. This recent approval is one of the many therapeutically novel strategies that are currently being exploited in clinical trials toward eventual contribution to the oncologist's toolbox against TNBC. In this review, we comprehensively discuss TNBC's distinct TME and its immunophenotype. Furthermore, we highlight the histological and molecular classification of this cancer. More importantly, we describe how these characteristics and classifications contribute to the current standards of care and how they steer the development of newer and more targeted therapies toward achieving peak therapeutic goals in the treatment of TNBC.

Anti-PD-1 and oncolytic viruses (OVs) have non-overlapping anti-tumor mechanisms, since each agent works at different steps of the cancer-immunity cycle. Evidence suggests that OVs improve therapeutic responses to anti-PD-1 therapy by reversing immunosuppressive factors, increasing the number and diversity of infiltrating lymphocytes, and promoting PD-L1 expression in both injected and non-injected tumors. Many studies in preclinical models suggest that the timing of anti-PD-1 administration influences the therapeutic success of the combination therapy (anti-PD-1 + OV). Therefore, determining the appropriate sequencing of agents is of critical importance to designing a rationale OV-based combinational clinical trial. Currently, the combination of anti-PD-1 and OVs are being delivered using various schedules, and we have classified the timing of administration of anti-PD-1 and OVs into five categories: (i) anti-PD-1 lead-in → OV; (ii) concurrent administration; (iii) OV lead-in → anti-PD-1; (iv) concurrent therapy lead-in → anti-PD-1; and (v) OV lead-in → concurrent therapy. Based on the reported preclinical and clinical literature, the most promising treatment strategy to date is hypothesized to be OV lead-in → concurrent therapy. In the OV lead-in → concurrent therapy approach, initial OV treatment results in T cell priming and infiltration into tumors and an immunologically hot tumor microenvironment (TME), which can be counterbalanced by engagement of PD-L1 to PD-1 receptor on immune cells, leading to T cell exhaustion. Therefore, after initial OV therapy, concurrent use of both OV and anti-PD-1 is critical through which OV maintains T cell priming and an immunologically hot TME, whereas PD-1 blockade helps to overcome PD-L1/PD-1-mediated T cell exhaustion. It is important to note that the hypothetical conclusion drawn in this review is based on thorough literature review on current understanding of OV + anti-PD-1 combination therapies and rhythm of treatment-induced cancer-immunity cycle. A variety of confounding factors such as tumor types, OV types, presence or absence of cytokine transgenes carried by an OV, timing of treatment initiation, varying dosages and treatment frequencies/duration of OV and anti-PD-1, etc. may affect the validity of our conclusion that will need to be further examined by future research (such as side-by-side comparative studies using all five treatment schedules in a given tumor model).

Syngeneic mouse tumor models are critical to investigating new immunotherapeutic modalities and providing a rationale for clinical translation. Oncolytic herpes simplex virus (oHSV) is an emerging treatment strategy for breast cancer. Genetically modified oHSV selectively replicates in cancer cells (sparring healthy cells), induces immunogenic cancer cell death, and stimulates innate/adaptive anti-tumor immune responses. The therapeutic success of oHSV depends on a dynamic interaction between oHSV, the tumor, and the host. Here, we evaluated the anti-tumor effects of G47Δ-IL12, a genetically engineered oHSV expressing interleukin-12 (IL-12), in two orthotopic immunocompetent mouse models of breast cancer, E0771 (some classified this model as luminal B subtype and some as triple-negative) and EMT6 (triple-negative subtype), which are syngeneic to C57BL/6 and BALB/c mice, respectively. Intratumoral G47Δ-IL12 treatment led to complete eradication of orthotopic E0771 and EMT6 mammary tumors and prevention of lung metastases, resulting in 100% long-term survivors, which remained tumor-free during the observation period (>100 days following the last G47Δ-IL12 treatment). In a contralateral orthotopic mammary tumor model, G47Δ-IL12 treatment of primary tumors led to the eradication of both treated and untreated distal tumors, confirming the abscopal and anti-metastatic effect of G47Δ-IL12 virotherapy. All cured mice from G47Δ-IL12-treated groups rejected lethal tumor re-challenge in the contralateral naïve mammary fat pad, indicating successful generation of a protective memory response. In both E0771 and EMT6 models, cell-specific IFN-γ responses and the presence of effector memory T cells (CD4+CD44highCD62Llow phenotype) in spleens of long-term survivors, possibly contributing to tumor relapse prevention. G47Δ-IL12-mediated anti-tumor efficacy was associated with significant increases in CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) (vs. mock group) in both E0771 and EMT6 tumor lesions. G47Δ-IL12 monotherapy also led to a significantly increased CD8+/FoxP3+ ratio (an important parameter for immunotherapy success) in both tumor models. Interestingly, immune cell depletion studies demonstrated that efficacy requires both CD4+ and CD8+ T cells in the E0771 model while only CD8+ T cells were required in the EMT6 model. Conclusions: G47Δ-IL12 monotherapy was effective in eradicating both orthotopic and metastatic tumors and generating long-term protective memory responses in two syngeneic breast cancer models. Treatment efficacy of G47Δ-IL12 was associated with significant immunologic alterations of the TME. G47Δ-IL12 utilized distinct immunologic mechanisms of action to eradicate tumors: CD8+ T cell-dependent action in the EMT6 model, whereas CD4+ and CD8+ T cell-dependent efficacy in the E0771 model. Altogether, our data suggest that G47Δ-IL12 is an efficient inflammatory modulator and beneficially exploits the immune system to eradicate breast tumors. These studies provide the necessary supporting evidence for the clinical translation of this agent into breast cancer patients. Citation Format: Hong-My Nguyen, Othon Almanza, Dipongkor Saha. Oncolytic herpes simplex virus immunovirotherapy eradicates breast cancer and prevents tumor relapse [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-11-01.

CD105 (endoglin) is a transmembrane protein that functions as a TGF-beta coreceptor and is highly expressed on endothelial cells. Unsurprisingly, preclinical and clinical evidence strongly suggests that CD105 is an important contributor to tumor angiogenesis and tumor progression. Emerging evidence suggests that CD105 is also expressed by tumor cells themselves in certain cancers such as renal cell carcinoma (RCC). In human RCC tumor cells, CD105 expression is associated with stem cell-like properties and contributes to the malignant phenotype in vitro and in xenograft models. However, as a regulator of TGF-beta signaling, there is a striking lack of evidence for the role of tumor-expressed CD105 in the anti-tumor immune response and the tumor microenvironment. In this study, we report that tumor cell-expressed CD105 potentiates both the in vitro and in vivo tumorigenic potential of RCC in a syngeneic murine RCC tumor model. Importantly, we find that tumor cell-expressed CD105 sculpts the tumor microenvironment by enhancing the recruitment of immunosuppressive cell types and inhibiting the polyfunctionality of tumor-infiltrating CD4

Renal cell carcinoma (RCC) is the deadliest form of urological cancer and is projected to be the fourth most common neoplasm in the USA in males by 2040. In addition to the current poor prognosis with 5-year survival rates hardly reaching 15%, the prevalence of resistance to currently available systemic therapies has also established an urgent need to develop new treatment regimen(s) for advanced RCC. Interferon-stimulated gene 15 (ISG15) is the first identified ubiquitin-like modifier and has been intensively studied for its central role in innate immunity against intracellular pathogens. However, in this study, we identified ISG15 as a novel tumor-associated antigen and prognostic marker in RCC. Further, we therapeutically targeted elevated ISG15 expression by means of a Listeria monocytogenes (Lm)-based vaccine, designated Lm-LLO-ISG15, in both subcutaneous and orthotopic RCC mouse models. Treatment with Lm-LLO-ISG15 resulted in an influx of tumor-infiltrating effector T cells and significant anti-tumor efficacy in both subcutaneous and orthotopic RCC tumor models. Treatment with Lm-LLO-ISG15 also generated a robust interferon-gamma response and attracted a larger pool of polyfunctional T cells into the tumor microenvironment. Importantly, the therapeutic efficacy of Lm-LLO-ISG15 in RCC is comparable to that of anti-PD-1 and sunitinib, the current frontline therapies for RCC patients. Collectively, our work illustrates that targeting ISG15 in RCC with a CTL-based immunotherapy such as Lm-LLO-ISG15 is a promising and potentially translatable therapeutic strategy to enhance survival in RCC patients.

Background: Colorectal cancer (CRC) has been a difficult-to-treat cancer with a significant proportion of microsatellite stable (MSS-CRC) patients who do not respond to immunotherapy. Currently, various strategies are being investigated to sensitize MSS-CRC tumors by converting them to immunologically “hot” tumors and improve responsiveness to immunotherapy. In this study, we demonstrate a novel strategy of combining a repurposed FDA-approved compound, Pimavanserin tartrate (PVT), with a Listeria monocytogenes (Lm)-based vaccine targeting Interferon-Stimulated Gene 15(ISG15), Lm-LLO-ISG15, against MSS-CRC tumors. Our findings demonstrate that both agents synergize to produce CD8+ T cell-dependent therapeutic efficacy in immunotherapy-resistant MSS-CRC tumors. Methods: The direct killing effect of PVT in vitro was confirmed by cytotoxic SRB assay. BALB/c mice bearing CT26, a murine model of MSS-CRC, were randomly assigned to receive either PBS, PVT, Lm-LLO-ISG15, or PVT+Lm-LLO-ISG15. Mice were monitored for tumor growth kinetics to study the anti-cancer effect of each monotherapy and the combination therapy. Changes in immune-related gene and protein expression were analyzed by qPCR and Western blot, respectively. Various immune cell subsets in the tumors and spleens were evaluated by multi-color flow cytometry and immunohistochemistry. In addition, organs such as the liver, kidney, heart, lungs, spleen, brain, and pancreas were collected for safety analysis. Results: PVT efficiently induced apoptosis and directly killed both human and mouse MSS-CRC cell lines in vitro. Further, PVT treatment enhanced T-cell activation and induced ISG15 expression, thus, sensitizing the tumor cells to targeting by the CTL-mediated immunotherapy, Lm-LLO-ISG15. While each monotherapy failed to demonstrate anti-tumor efficacy, treatment with the combination therapy (PVT + Lm-LLO-ISG15) significantly controlled tumor burden and extended the median survival. The therapeutic efficacy of the combination approach was associated with a higher infiltration of CD8+ T cells in the TME and an increased population of effector memory T cells (CD4+CD44hiCD62Llo) in the spleen. The anti-tumor efficacy of combination therapy on subcutaneous CT26 tumor growth was completely abrogated after the depletion of CD8+ T cells. Moreover, the safety profile of dual therapy was comparable to that of each monotherapy and PBS. Conclusions: MSS-CRCs are considered “immunologically cold” tumors that represent great challenges for any standalone therapies. In this study, we evaluated, for the first time, the therapeutic efficacy of a novel combination of PVT and Lm-LLO-ISG15 in an aggressive MSS-CRC mouse model. Combination therapy demonstrated a synergistic effect that led to a significant reduction of tumor burden and an extension of median survival. While the underlying mechanism for synergism requires further examination, our findings strongly suggest that combining PVT and Lm-LLO-ISG15 could be a promising approach for MSS-CRC patients. Citation Format: Shreyas Ramchandra Gaikwad, Hong My Nguyen, Sanjay K. Srivastava, Laurence Wood. A novel combination of Listeria-based immunotherapy and repurposed drug combats colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB201.

Glioblastoma (GBM) is a lethal primary malignant brain tumor with no current effective treatments. The recent emergence of immuno-virotherapy and FDA approval of T-VEC have generated a great expectation towards oncolytic herpes simplex viruses (oHSVs) as a promising treatment option for GBM. Since the generation and testing of the first genetically engineered oHSV in glioma in the early 1990s, oHSV-based therapies have shown a long way of great progress in terms of anti-GBM efficacy and safety, both preclinically and clinically. Here, we revisit the literature to understand the recent advancement of oHSV in the treatment of GBM. In addition, we discuss current obstacles to oHSV-based therapies and possible strategies to overcome these pitfalls.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in both men and women in the United States. While immune checkpoint inhibitor (ICI) therapy is demonstrating remarkable clinical responses, the resistance and immune-related toxicities associated with ICIs demonstrate the need to develop additional immunotherapy options for CRC patients. Cancer vaccines represent a safe and promising treatment approach for CRC. As previously developed tumor-associated antigen (TAA)-based cancer vaccines for CRC are not demonstrating promising results, we propose that interferon-stimulated gene 15 (ISG15) is a novel TAA and therapeutic target for CRC. Our work demonstrates the anti-tumor efficacy of a Listeria-based vaccine targeting ISG15, designated Lm-LLO-ISG15, in an immunocompetent CRC murine model. The Lm-LLO-ISG15-mediated anti-tumor response is associated with an increased influx of functional T cells, higher production of multiple intracellular cytokines response, a lower number of regulatory T cells, and a greater ratio of effector to regulatory T cells (Teff/Treg) in the tumor microenvironment.

Oncolytic viruses (OVs), such as the oncolytic herpes simplex virus (oHSV), are a rapidly growing treatment strategy in the field of cancer immunotherapy. OVs, including oHSV, selectively replicate in and kill cancer cells (sparing healthy/normal cells) while inducing anti-tumor immunity. Because of these unique properties, oHSV-based treatment strategies are being increasingly used for the treatment of cancer, preclinically and clinically, including FDA-approved talimogene laherparevec (T-Vec). Growth, purification, and titration are three essential laboratory techniques for any OVs, including oHSVs, before they can be utilized for experimental studies. This paper describes a simple step-by-step method to amplify oHSV in Vero cells. As oHSVs multiply, they produce a cytopathic effect (CPE) in Vero cells. Once 90-100% of the infected cells show a CPE, they are gently harvested, treated with benzonase and magnesium chloride (MgCl2), filtered, and subjected to purification using the sucrose-gradient method. Following purification, the number of infectious oHSV (designated as plaque-forming units or PFUs) is determined by a "plaque assay" in Vero cells. The protocol described herein can be used to prepare high-titer oHSV stock for in vitro studies in cell culture and in vivo animal experiments.

Hong-My Nguyen, Dipongkor Saha Department of Immunotherapeutics and Biotechnology, Texas Tech University Health Sciences Center, School of Pharmacy, Abilene, TX, 79601, USACorrespondence: Dipongkor Saha Tel +1 325-6960583Email Dipongkor.saha@ttuhsc.eduAbstract: Glioblastoma (GBM) is a lethal primary malignant brain tumor with no current effective treatments. The recent emergence of immuno-virotherapy and FDA approval of T-VEC have generated a great expectation towards oncolytic herpes simplex viruses (oHSVs) as a promising treatment option for GBM. Since the generation and testing of the first genetically engineered oHSV in glioma in the early 1990s, oHSV-based therapies have shown a long way of great progress in terms of anti-GBM efficacy and safety, both preclinically and clinically. Here, we revisit the literature to understand the recent advancement of oHSV in the treatment of GBM. In addition, we discuss current obstacles to oHSV-based therapies and possible strategies to overcome these pitfalls.Keywords: glioblastoma, oncolytic herpes simplex virus, armed oHSV, re-targeted oHSV, combination therapy

Simple Summary Glioblastoma (GBM) is the most common form of primary malignant brain tumor with a devastatingly poor prognosis. Tumor heterogeneity (cellular, molecular and immune) is the major obstacle to current treatment failure. We revisited the recent literature to understand the heterogeneous features of GBM and their potential role in treatment resistance. This review provides a comprehensive overview covering the GBM’s pathogenetic features, currently available treatment options and the treatments currently under development in the clinic. Abstract Glioblastoma (GBM) is the most common form of primary malignant brain tumor with a devastatingly poor prognosis. The disease does not discriminate, affecting adults and children of both sexes, and has an average overall survival of 12–15 months, despite advances in diagnosis and rigorous treatment with chemotherapy, radiation therapy, and surgical resection. In addition, most survivors will eventually experience tumor recurrence that only imparts survival of a few months. GBM is highly heterogenous, invasive, vascularized, and almost always inaccessible for treatment. Based on all these outstanding obstacles, there have been tremendous efforts to develop alternative treatment options that allow for more efficient targeting of the tumor including small molecule drugs and immunotherapies. A number of other strategies in development include therapies based on nanoparticles, light, extracellular vesicles, and micro-RNA, and vessel co-option. Advances in these potential approaches shed a promising outlook on the future of GBM treatment. In this review, we briefly discuss the current understanding of adult GBM’s pathogenetic features that promote treatment resistance. We also outline novel and promising targeted agents currently under development for GBM patients during the last few years with their current clinical status.

Triple-negative breast cancer (TNBC) is a difficult-to-treat disease with high rates of local recurrence, distant metastasis, and poor overall survival with existing therapies. Thus, there is an unmet medical need to develop new treatment regimen(s) for TNBC patients. An oncolytic herpes simplex virus encoding a master anti-tumor cytokine, interleukin 12, (designated G47Δ-mIL12) selectively kills cancer cells while inducing anti-tumor immunity. G47Δ-mIL12 efficiently infected and killed murine (4T1 and EMT6) and human (HCC1806 and MDA-MB-468) mammary tumor cells in vitro. In vivo in the 4T1 syngeneic TNBC model, it significantly reduced primary tumor burden and metastasis, both at early and late stages of tumor development. The virus-induced local and abscopal effects were confirmed by significantly increased infiltration of CD45+ leukocytes and CD8+ T cells, and reduction of granulocytic and monocytic MDSCs in tumors, both treated and untreated contralateral, and in the spleen. Significant trafficking of dendritic cells (DCs) were only observed in spleens of virus-treatment group, indicating that DCs are primed and activated in the tumor-microenvironment following virotherapy, and trafficked to lymphoid organs for activation of immune cells, such as CD8+ T cells. DC priming/activation could be associated with virally enhanced expression of several antigen processing/presentation genes in the tumor microenvironment, as confirmed by NanoString gene expression analysis. Besides DC activation/priming, G47Δ-mIL12 treatment led to up-regulation of CD8+ T cell activation markers in the tumor microenvironment and inhibition of tumor angiogenesis. The anti-tumor effects of G47Δ-mIL12 treatment were CD8-dependent. These studies illustrate the ability of G47Δ-mIL12 to immunotherapeutically treat TNBC.

Oncolytic viruses (OVs) are genetically modified or naturally occurring viruses, which preferentially replicate in and kill cancer cells while sparing healthy cells, and induce anti-tumor immunity. OV-induced tumor immunity can be enhanced through viral expression of anti-tumor cytokines such as interleukin 12 (IL-12). IL-12 is a potent anti-cancer agent that promotes T-helper 1 (Th1) differentiation, facilitates T-cell-mediated killing of cancer cells, and inhibits tumor angiogenesis. Despite success in preclinical models, systemic IL-12 therapy is associated with significant toxicity in humans. Therefore, to utilize the therapeutic potential of IL-12 in OV-based cancer therapy, 25 different IL-12 expressing OVs (OV-IL12s) have been genetically engineered for local IL-12 production and tested preclinically in various cancer models. Among OV-IL12s, oncolytic herpes simplex virus encoding IL-12 (OHSV-IL12) is the furthest along in the clinic. IL-12 expression locally in the tumors avoids systemic toxicity while inducing an efficient anti-tumor immunity and synergizes with anti-angiogenic drugs or immunomodulators without compromising safety. Despite the rapidly rising interest, there are no current reviews on OV-IL12s that exploit their potential efficacy and safety to translate into human subjects. In this article, we will discuss safety, tumor-specificity, and anti-tumor immune/anti-angiogenic effects of OHSV-IL12 as mono- and combination-therapies. In addition to OHSV-IL12 viruses, we will also review other IL-12-expressing OVs and their application in cancer therapy.

A chitin wound healing membrane was extracted and minimally processed from soft-shell crab carapace, a fabrication method simpler than the conventional process of chemically modifying chitin into more soluble and bioactive derivatives. We observed an interesting difference between the topography of the membrane’s internal and external surface, which correlated with their different abilities to promote cell adhesion and proliferation. The internal surface was dotted with prickles where cells could adhere and elongate, while the smooth external surface demonstrated poor cell adhesion and proliferation. Applying the internal surface of the membrane onto the wound improved the epidermis surface homogeneity and accelerated epidermis-dermis re-attachment. This membrane also showed an excellent tensile strength of 105.7 ± 29.9 MPa and ultimate strain of 6.5 ± 4.2%, higher than those of common wound healing membranes from cellulose, and collagen. These results showed the ability of chitin membranes from soft-shell crab carapaces to support cell attachment, proliferation, and migration and its potential for further wound healing applications.

International audience