Search

Tumor recurrence after discontinuation of cabozantinib treatment.

Resistance to currently available targeted therapies significantly hampers the survival of patients with prostate cancer with bone metastasis. Here we demonstrate an important resistance mechanism initiated from tumor-induced bone. Studies using an osteogenic patient-derived xenograft, MDA-PCa-118b, revealed that tumor cells resistant to cabozantinib, a Met and VEGFR-2 inhibitor, reside in a “resistance niche” adjacent to prostate cancer-induced bone. We performed secretome analysis of the conditioned medium from tumor-induced bone to identify proteins (termed “osteocrines”) found within this resistance niche. In accordance with previous reports demonstrating that activation of integrin signaling pathways confers therapeutic resistance, 27 of the 90 osteocrines identified were integrin ligands. We found that following cabozantinib treatment, only tumor cells positioned adjacent to the newly formed woven bone remained viable and expressed high levels of pFAK-Y397 and pTalin-S425, mediators of integrin signaling. Accordingly, treatment of C4-2B4 cells with integrin ligands resulted in increased pFAK-Y397 expression and cell survival, whereas targeting integrins with FAK inhibitors PF-562271 or defactinib inhibited FAK phosphorylation and reduced the survival of PC3-mm2 cells. Moreover, treatment of MDA-PCa-118b tumors with PF-562271 led to decreased tumor growth, irrespective of initial tumor size. Finally, we show that upon treatment cessation, the combination of PF-562271 and cabozantinib delayed tumor recurrence in contrast to cabozantinib treatment alone. Our studies suggest that identifying paracrine de novo resistance mechanisms may significantly contribute to the generation of a broader set of potent therapeutic tools that act combinatorially to inhibit metastatic prostate cancer. Cancer Res; 75(22); 4949–59. ©2015 AACR.

Primers used in PCR.

Cancer vaccines harness the host immune system to generate antigen-specific antitumor immunity for long-term tumor elimination with durable immunomodulation. Commonly investigated strategies reintroduce ex vivo autologous dendritic cells (DCs) but have limited clinical adoption due to difficulty in manufacturing, delivery and low clinical efficacy. To combat this, we designed the “NanoLymph”, an implantable subcutaneous device for antigen-specific antitumor immunomodulation. The NanoLymph consists of a dual-reservoir platform for sustained release of immune stimulants via a nanoporous membrane and hydrogel-encapsulated antigens for local immune cell recruitment and activation, respectively. Here, we present the development and characterization of the NanoLymph as well as efficacy validation for immunomodulation in an immunocompetent murine model. Specifically, we established the NanoLymph biocompatibility and mechanical stability. Further, we demonstrated minimally invasive transcutaneous refilling of the drug reservoir in vivo for prolonging drug release duration. Importantly, our study demonstrated that local elution of two drugs (GMCSF and Resiquimod) generates an immune stimulatory microenvironment capable of local DC recruitment and activation and generation of antigen-specific T lymphocytes within 14 days. In summary, the NanoLymph approach can achieve in situ immunomodulation, presenting a viable strategy for therapeutic cancer vaccines.

Although head and neck squamous cell carcinoma (HNSCC) has in the past been largely associated with tobacco use, human papillomavirus (HPV+) oropharynx cancer has in recent years emerged as the fastest growing type of HNSCC. Patients with HPV+ HNSCC have a better prognosis; however, the 5-year survival for both HPV+ and HPV- subtypes with recurrent or metastatic disease is poor. To gain insights into the tumor microenvironments of both HNSCC subtypes and identify potential therapeutic targets, we performed epigenomic deconvolution on 580 HNSCC samples from the TCGA dataset. Deconvolution revealed distinct molecular and histoepigenetic profiles of the two tumor subtypes, including their cellular composition, epigenomic profiles and gene expression for constituent cell types, and potential cancer cell-specific targets. Our analyses show that high abundance of both CD8 T-cells and B-cells explains better prognosis in HPV+ HNSCC. Deconvolution of gene expression profiles revealed higher expression of the immunotherapy target PD-1 in HPV+ immune cells compared to HPV- cells, suggesting that HPV+ tumors may preferentially benefit from PD-1 targeted therapy. Further analyses identified HPV+ and HPV- cancer cell surface proteins that can also serve as potential targets for therapy. Specifically, Wnt pathway receptor ROR2 is preferentially overexpressed in HPV+ subtypes, suggesting opportunities for development of targeted therapy based on HPV status. In summary, the comprehensive molecular and histoepigenetic analysis of tumor microenvironments by epigenomic deconvolution reveals potential novel biomarkers and targets for precision therapy of HNSCC.

Hypertension is a chronic condition that requires lifelong therapeutic management. Strict adherence to drug administration timing improves efficacy, while poor adherence leads to safety concerns. In light of these challenges, we present a nanofluidic technology that enables long-acting drug delivery with tunable timing of drug administration using buried gate electrodes in nanochannels. We developed a Poly(ethylene glycol) methyl ether-block-poly(ε-caprolactone) (PEG-PCL)-based micellar formulation of amlodipine besylate, a calcium channel blocker for hypertension treatment. The electrostatically charged PEG-PCL micellar formulation enhanced drug solubility and rendered amlodipine responsive to electrostatic release gating in nanochannels for sustained release at clinically relevant therapeutic dose. Using a low-power (

Landmark successes in oncoimmunology have led to development of therapeutics boosting the host immune system to eradicate local and distant tumors with impactful tumor reduction in a subset of patients. However, current immunotherapy modalities often demonstrate limited success when involving immunologically cold tumors and solid tumors. Here, we describe the role of various biomaterials to formulate cancer vaccines as a form of cancer immunotherapy, seeking to utilize the host immune system to activate and expand tumor-specific T cells. Biomaterial-based cancer vaccines enhance the cancer-immunity cycle by harnessing cellular recruitment and activation against tumor-specific antigens. In this review, we discuss biomaterial-based vaccine strategies to induce lymphocytic responses necessary to mediate anti-tumor immunity. We focus on strategies that selectively attract dendritic cells via immunostimulatory gradients, activate them against presented tumor-specific antigens, and induce effective cross-presentation to T cells in secondary lymphoid organs, thereby generating immunity. We posit that personalized cancer vaccines are promising targets to generate long-term systemic immunity against patient- and tumor-specific antigens to ensure long-term cancer remission.

Background Pancreatic adenocarcinoma (PDAC) is associated with extremely poor prognosis and a 5-year survival rate of 10% and remains a lethal malignancy. Surgical resection and combination with chemoradiotherapy are the current standard-of-care options, may improve long-term survival in localized disease; however, the majority of patients are diagnosed at advanced stage. The incorporation of immunotherapy in the treatment algorithm convenes a new era for PDAC treatment. Several immunotherapy approaches have been investigating for treating PDAC such as checkpoint inhibitors, vaccines, adoptive cell therapy, and so on. Immunotherapy has been shown as a promising therapeutic method for many cancer types; however, the complexity and immunosuppressive of the solid tumor microenvironment (TME) results in limited treatment efficacy for PDAC. Methods To sensitize the TME in response to immunotherapy, we developed an implantable intratumoral drug delivery device, Nanofluidic Drug-Eluting Seed (NDES) can be injected via a minimally invasive trocar system that feasible for the clinical setting. NDES has shown efficiently delivered immunotherapy to murine breast cancer model and reduced tumor burden and showed low liver inflammation compared to the intraperitoneal delivery approach in the previous study.1 2 Here, we utilized NDES for the sustained intratumoral delivery of the CD40 antibody. We compared the efficacy of NDES against intraperitoneal and intratumoral administration, which mimics conventional systemic treatment. Tumor growth was investigated for treatment efficacy. Local and systemic immune responses were assessed via flow cytometry. Results NDES delivered CD40 significantly reduced tumor burden, some even achieved tumor clearance. Local NDES CD40 delivery approach showed a systemic increase of CD8+ and CD4+ T cells in the tumor-draining lymph node and spleen by flow cytometry. Furthermore, NDES CD40 treated mice showed an increase of CD8+ and CD4+ central memory T cells locally and systemically. We also investigated the combination with radiotherapy, no significant difference in tumor burden was observed when compared to single-agent CD40 antibody. The results indicated CD40 promotes TME response and improved treatment efficacy. Conclusions These immunological responses demonstrate ‘cold’ to ‘hot’ tumor transformation, which translated to tumor burden reduction. Overall, NDES delivery strategy offers promise for enhancing therapeutic index and transforming the landscape of PDAC tumor therapy. References Liu H-C, Viswanath D, Pesaresi F, et al. Potentiating antitumor efficacy through radiation and sustained intratumoral delivery of anti-CD40 and anti-PDL1. Int J Radiat Oncol Biol Phys 2020;S0360-3016(20)33745-7. Chua CYX, Jain P, et al. Nanofluidic drug-eluting seed for sustained intratumoral immunotherapy in triple negative breast cancer. J Control Release 2018;285:23–34.

Background Personalized therapeutic cancer vaccines aim to target and reprogram the host immune system to achieve cancer eradication in situ. Cancer vaccines deliver two main components: immunostimulants (iS) and tumor antigens to reduce tumor burden with a robust T cell response; however, none have reached broad clinical success due to difficulty in vaccine administration, ex vivo cellular manipulation, low clinical efficacy and broad administrative barriers. While most efforts to date have focused on repeated bolus administrations, biomaterial-based vaccine strategies have led to promising clinical translation. Methods In light of these challenges, we have designed a clinically-viable platform-based vaccine strategy, termed the NanoLymph, to provide spatiotemporal elution of immunostimulants and tumor antigens locally to recruit and activate antitumor immunity for cancer eradication. Here, we aim to target the release of granulocyte macrophage colony stimulating factor (GM-CSF) and TLR-7/8 agonist Resiquimod (R848) to promote recruitment and activation of dendritic cells (DCs), a key player in antitumor cytotoxicity. Results We demonstrate the NanoLymph as an structurally stable and biocompatible immunostimulatory niche for durable DC-driven tumor specific T-cell mediated cytotoxicity. Additionally, we demonstrate the NanoLymph’s ability to recruit and activate immune cells of interest, activating antitumor immunity against model antigen. Thus, we have provided the framework necessary to develop a personalized therapeutic cancer vaccine for tumor-specific T-cell mediated responses necessary to generate immunological memory. Conclusions Future studies will evaluate immunostimulant and tumor antigen biodistribution in vivo and further apply the NanoLymph in a tumor bearing model to effect antitumor cytotoxicity. Ultimately, we aim to develop a personalized platform applicable for every patient of any cancer type aimed at direct clinical translation. Ethics Approval This study was approved by the Houston Methodist Research Institute (HMRI), according to protocols approved by the Institutional Animal Care and Use Committee (IACUC). HMRI’s Animal Welfare Assurance number is A4555-01. HMRI assures strict compliance with all federal regulations and guidelines involving the use of laboratory animals in biomedical research.

Carbon fibers reinforced polymers (CFRPs) are prolifically finding applications in the medical field, moving beyond the aerospace and automotive industries. Owing to its high strength-to-weight ratio, lightness and radiolucency, CFRP-based materials are emerging to replace traditional metal-based medical implants. Numerous types of polymers matrices can be incorporated with carbon fiber using various manufacturing methods, creating composites with distinct properties. Thus, prior to biomedical application, comprehensive evaluation of material properties, biocompatibility and safety are of paramount importance. In this study, we systematically evaluated a series of novel CFRPs, aiming at analyzing biocompatibility for future development into medical implants or implantable drug delivery systems. These CFRPs were produced either via Carbon Fiber-Sheet Molding Compound or Fused Deposition Modelling-based additive manufacturing. Unlike conventional methods, both fabrication processes afford high production rates in a time-and cost-effective manner. Importantly, they offer rapid prototyping and customization in view of personalized medical devices. Here, we investigate the physicochemical and surface properties, material mutagenicity or cytotoxicity of 20 CFRPs, inclusive of 2 surface finishes, as well as acute and sub-chronic toxicity in mice and rabbits, respectively. We demonstrate that despite moderate in vitro physicochemical and surface changes over time, most of the CFRPs were non-mutagenic and non-cytotoxic, as well as biocompatible in small animal models. Future work will entail extensive material assessment in the context of orthopedic applications such as evaluating potential for osseointegration, and a chronic toxicity study in a larger animal model, pigs.

Purpose Mounting evidence demonstrates that combining radiation therapy (RT) with immunotherapy can reduce tumor burden in a subset of patients. However, conventional systemic delivery of immunotherapeutics is often associated with significant adverse effects, which force treatment cessation. The aim of this study was to investigate a minimally invasive therapeutics delivery approach to improve clinical response while attenuating toxicity. Methods and Materials We used a nanofluidic drug-eluting seed (NDES) for sustained intratumoral delivery of combinational antibodies CD40 and PDL1. To enhance immune and tumor response, we combined the NDES intratumoral platform with RT to treat the 4T1 murine model of advanced triple negative breast cancer. We compared the efficacy of NDES against intraperitoneal administration, which mimics conventional systemic treatment. Tumor growth was recorded, and local and systemic immune responses were assessed via imaging mass cytometry and flow cytometry. Livers and lungs were histologically analyzed for evaluation of toxicity and metastasis, respectively. Results The combination of RT and sustained intratumoral immunotherapy delivery of CD40 and PDL1 via NDES (NDES CD40/PDL1) showed an increase in both local and systemic immune response. In combination with RT, NDES CD40/PDL1 achieved significant tumor burden reduction and liver inflammation mitigation compared with systemic treatment. Importantly, our treatment strategy boosted the abscopal effect toward attenuating lung metastatic burden. Conclusions Overall, our study demonstrated superior efficacy of combination treatment with RT and sustained intratumoral immunotherapy via NDES, offering promise for improving therapeutic index and clinical response.

Probiotics exert multiple health benefits, including gastrointestinal health, immunoregulation, and metabolic disease improvement, by modulating microbiota to maintain eubiosis via the hypothalamic&ndash, pituitary&ndash, adrenal (HPA) and brain&ndash, gut&ndash, microbiome axes. Physiological fatigue, mental stress, and gastrointestinal discomfort under the demands of athletic performance as well as immunosuppression are common during endurance training and competition. Limited studies investigated the functional effects of probiotic supplementation on endurance training. Bifidobacterium longum subsp. Longum OLP-01 (OLP-01), isolated from an elite Olympic athlete, was combined with a six-week exercise training program with gradually increasing intensity. In this study, Institute of Cancer Research (ICR) mice were assigned to sedentary, exercise, OLP-01, or exercise + OLP-01 groups and administered probiotic and/or treadmill exercise training for six weeks to assess exercise performance, physiological adaption, and related metabolites. The exercise + OLP-01 group demonstrated higher performance in terms of endurance and grip strength, as well as improved fatigue-associated indexes (lactate, ammonia, creatine kinase (CK), lactate dehydrogenase (LDH), and glycogen content), compared with the other groups. OLP-01 supplementation significantly ameliorated inflammation and injury indexes (platelet/lymphocyte ratio (PLR), aminotransferase (AST), and CK) caused by prolonged endurance exercise test. Moreover, acetate, propionate, and butyrate levels were significantly higher in the exercise + OLP-01 group than in the sedentary and OLP-01 groups. Athletes often experience psychological and physiological stress caused by programed intensive exercise, competition, and off-site training, often leading to poor exercise performance and gastrointestinal issues. Functional OLP-01 probiotics are considered to be a nutritional strategy for improving physiological adaption, oxidative stress, inflammation, and energy balance to ensure high physical performance. Based on these results, probiotics combined with exercise training is a potential strategy for ensuring high physical performance of athletes, which should be further investigated through microbiota validation.

Cell encapsulation is an attractive transplantation strategy to treat endocrine disorders. Transplanted cells offer a dynamic and stimulus-responsive system that secretes therapeutics based on patient need. Despite significant advancements, a challenge in allogeneic cell encapsulation is maintaining sufficient oxygen and nutrient exchange, while providing protection from the host immune system. To this end, we developed a subcutaneously implantable dual-reservoir encapsulation system integrating in situ prevascularization and local immunosuppressant delivery, termed NICHE. NICHE structure is 3D-printed in biocompatible polyamide 2200 and comprises of independent cell and drug reservoirs separated by a nanoporous membrane for sustained local release of immunosuppressant. Here we present the development and characterization of NICHE, as well as efficacy validation for allogeneic cell transplantation in an immunocompetent rat model. We established biocompatibility and mechanical stability of NICHE. Further, NICHE vascularization was achieved with the aid of mesenchymal stem cells. Our study demonstrated sustained local elution of immunosuppressant (CTLA4Ig) into the cell reservoir protected transcutaneously-transplanted allogeneic Leydig cells from host immune destruction during a 31-day study, and reduced systemic drug exposure by 12-fold. In summary, NICHE is the first encapsulation platform achieving both in situ vascularization and immunosuppressant delivery, presenting a viable strategy for allogeneic cell transplantation.

Figure S2. Individual tumor growth curves for singlet and dual treatments and CTX/L-NIL gene expression. Subcutaneous established mEER tumors (day 17–18 post tumor cell injection) were treated with individual or dual treatment combinations of PD-1/CTLA-4, CTX/L-NIL, and radiation (RT) according to the same schedule shown in Figs. 1c and 2b. (A) Individual mEER tumor growth curves for 2 experiments, one of which was used for in Fig. 1d (N = 2; n = 7–17 per group). (B) Individual tumor growth curves for singlet and dual treatment combinations of CPR regimen (N = 2–3; n = 12–19). (C) Differential gene expression of CTX/L-NIL treated tumors compared to control tumors compared after 1 week (day 23) of treatment with PD-L1 and PD-L2 noted in red dots (N = 1; n = 9 per group). Blue lines indicate gene 2-fold change point (vertical) and corrected p-value less than 0.0001 (horizontal). (PDF 3329 kb)

Figure S12. Lymphoid flow cytometry gating strategies. Flow cytometry strategy used for gating lymphoid subsets, with labeled cell type above the respective gate. (A) T cell phenotype panel which included various markers to assess T cell memory, activation, and effector status. (B) T cell exhaustion panel which included markers to assess regulatory T cells as well as CD8+ and CD4+ T cell markers of exhaustion. (PDF 4584 kb)

Resistance to currently available targeted therapies significantly hampers the survival of patients with prostate cancer with bone metastasis. Here we demonstrate an important resistance mechanism initiated from tumor-induced bone. Studies using an osteogenic patient-derived xenograft, MDA-PCa-118b, revealed that tumor cells resistant to cabozantinib, a Met and VEGFR-2 inhibitor, reside in a “resistance niche” adjacent to prostate cancer-induced bone. We performed secretome analysis of the conditioned medium from tumor-induced bone to identify proteins (termed “osteocrines”) found within this resistance niche. In accordance with previous reports demonstrating that activation of integrin signaling pathways confers therapeutic resistance, 27 of the 90 osteocrines identified were integrin ligands. We found that following cabozantinib treatment, only tumor cells positioned adjacent to the newly formed woven bone remained viable and expressed high levels of pFAK-Y397 and pTalin-S425, mediators of integrin signaling. Accordingly, treatment of C4-2B4 cells with integrin ligands resulted in increased pFAK-Y397 expression and cell survival, whereas targeting integrins with FAK inhibitors PF-562271 or defactinib inhibited FAK phosphorylation and reduced the survival of PC3-mm2 cells. Moreover, treatment of MDA-PCa-118b tumors with PF-562271 led to decreased tumor growth, irrespective of initial tumor size. Finally, we show that upon treatment cessation, the combination of PF-562271 and cabozantinib delayed tumor recurrence in contrast to cabozantinib treatment alone. Our studies suggest that identifying paracrine de novo resistance mechanisms may significantly contribute to the generation of a broader set of potent therapeutic tools that act combinatorially to inhibit metastatic prostate cancer. Cancer Res; 75(22); 4949–59. ©2015 AACR.

HER2, commonly referred to as ErbB2, is a receptor tyrosine kinase that, along with EGFR, makes up one of the four members of the ErbB family of proteins. These ErbB proteins are expressed in most epithelial cell layers and play a key role in cell differentiation. HER2 has been found to be overexpressed in a number of human cancers, including breast and gastric carcinomas. Overexpression of the HER2 receptor represents a potential target for chimeric antigen receptor (CAR) T cell therapy. CAR T clinical trials in leukemia and lymphoma have demonstrated durable remission of the disease or even cure, but application of CAR T cells to solid tumors such as head and neck squamous cell carcinoma (HNSCC) lags behind. To evaluate HER2 expression in HNSCC, we prepared 4 different tumor microarrays (TMAs) of patient tumors based on their location (larynx, salivary gland, oropharynx or oral cavity) for IHC staining. These TMAs were then stained and scored by two different methods: with an anti-HER2 monoclonal antibody (CB11) and scoring based on breast cancer guidelines, or with the FDA-approved HER2 HercepTest IHC procedure. Based on the level of HER2 expression determined by TMA staining, we identify HNSCC as a candidate tumor type for preclinical testing of HER2-specific CAR T therapy. We utilize the chick embryo chorioallantoic membrane (CAM) tumor model, a naturally immune-deficient platform for growth of vascularized, three-dimensional tumors, as a rapid and inexpensive system to assess CAR T efficacy against human HNSCC tumors in vivo. To demonstrate tumor killing efficacy of HER2-specific CAR T cells (HER2.CAR Ts) on the CAM, we engrafted tumors derived from the HER2-positive (+) HNSCC cell line FaDu and HER2-negative (–) breast adenocarcinoma cell line MDA-MB-468 onto the CAM of day 7 fertilized chicken eggs. Both cell lines had been previously genetically engineered to express firefly luciferase (ffLuc). On day 10, established tumors were treated with HER2.CAR Ts generated by retroviral transduction of primary activated human T cells with a second-generation CAR T construct incorporating a CD28.ζ signaling domain. Four days after CAR T treatment, tumors were excised from the CAM, homogenized, and lysed; the luminescence of the resulting cell lysates (ffLuc activity) was used to quantify the relative number of viable tumor cells. While CAR T treatment resulted in an average 56% decrease in tumor size in the HER2+ FaDu tumors, there was no significant change in HER2– MDA-MB-468 tumor size. These results suggest that HER2-expressing HNSCC can be effectively targeted by HER2-directed CAR T calls and demonstrate the potential of the CAM tumor model as a cost-effective tool for rapid preclinical assessment of CAR T cell therapy. Citation Format: Emilie A. Warren, Hsuan-Chen Liu, Caroline E. Porter, Kershena S. Liao, Meenakshi Hegde, Wendong Yu, Patricia D. Castro, Vlad Sandulache, Nabil Ahmed, Masataka Suzuki, Andrew Sikora. Overexpression of HER2 in head and neck cancer represents a potential target for T cell immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 574.

Immune checkpoint inhibitors (ICI), including those targeting cytotoxic T-lymphocyte-associated-antigen-4 (CTLA-4) and programmed cell death receptor-1 (PD-1), have shown tremendous potential against solid tumor malignancies; however, response to ICI remains unpredictable with 60-90% of patients receiving minimal to no benefit. Lack of efficacy is commonly attributed to inadequate tumor-specific T cell generation and the immunosuppressive effects of the tumor immune microenvironment (TIME). Thus, we hypothesized that a combinatory treatment strategy aiming to enhance antigen presentation and revert the immunosuppressive TIME could improve response rates of ICI in established solid tumors. Using a syngeneic tumor model of HPV-associated head and neck cancer (mEER) established to 60-75 mm2 in size, we found that CTLA-4 and/or PD-1 inhibition only minorly delayed tumor growth and flow cytometry profiling revealed that the TIME maintained a “cold” or immunosuppressed state similar to untreated tumors. When PD-1/CTLA-4 inhibition was combined with a weekly dose of tumor-directed radiation (10 Gy x 2), we observed upregulation of antigen presentation molecules in the draining lymph node but the combination remained incapable of generating long-term survival benefit. This lack of efficacy was attributed to the immunosuppressed and lymphodepleted TIME, a common consequence of radiation. Thus, to improve the TIME, we optimized an immune-stimulating drug combination previously developed in our lab combining cyclophosphamide (CTX) and a small molecule inducible nitric oxide synthase (iNOS) inhibitor L-n6-(1-iminoethyl)-lysine (L-NIL). When we combined CTX/L-NIL immunomodulation, PD-1/CTLA-4 checkpoint inhibition, and radiation (collectively called the “CPR” regimen), we observed complete rejection of approximately 70% of established tumors in a CD8 T-cell dependent manner and potent development of immunologic memory against tumor-associated antigens. Tumor immune profiling after treatment revealed a “cold to hot” transition of the TIME, including increased levels of myeloid and lymphoid subsets associated with anti-tumoral immune responses (i.e. CD8 T cells, dendritic cells, M1 macrophages) and downregulation of immunosuppressive cellular subsets (i.e. T regulatory cells, granulocytic myeloid derived suppressor cells). We observed strong lymphoproliferation effects in the tumor-draining lymph node which resulted in significant TIME improvements including a 15-fold increase in the CD8 to regulatory T cell ratio. Thus, we have demonstrated that the rational combination of TIME immunomodulation, localized radiation to enhance antigen presentation, and immune checkpoint inhibitors to prevent T-cell exhaustion can promote the immunologic rejection of established solid tumors. Citation Format: Jared M. Newton, Aurelie Hanoteau, Hsuan-Chen Liu, Angelina Gaspero, Robyn D. Gartrell, Thomas D. Hart, Damya Laoui, Falguni Parikh, Yvonne M. Saenger, Andrew G. Sikora. Radiation, immune checkpoint inhibition, and modulation of the tumor immune microenvironment promotes immunologic rejection of established HPV-associated tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4068.

Human papillomavirus (HPV)-associated Head and neck cancer (HNSCC) are among the fastest growing cancer types. In contrast to non-viral HNSCC caused by traditional risk factors, tobacco and alcohol, HPV-HNSCC displayed distinct molecular alterations and prognosis from HPV-negative HNSCC. Currently, there is no clinically-approved therapeutic vaccine, and no targeted therapy approach for HPV-driven cancer which exploits the unique biology of HPV infection. In order to identify novel targets suitable for therapeutic and diagnostic development, we developed two parallel approaches to identification of proteins/antigens expressed on the surface of the host cells, whose expression is altered by HPV infection. One is an “antigen-agnostic” approach by using immortalized HNSCC cell membrane fractions to generate monoclonal antibodies, which does not require advance knowledge of the identities of target antigens. The second approach is bioinformatics analyses of the TCGA database using the epigenomic deconvolution tool (EDec) to identify surface proteins that are differentially overexpressed in HPV- HNSCC. Five thousand hybridoma colonies were generated by the “antigen-agnostic” approach, and were then screened by flow cytometry to test the specificity of binding to HPV-positive cancer cell lines (2 HNSCC and 2 Cervical Cancer) and HPV-negative cancer cell lines (4 HNSCC and 1 CC). After primary screening, we narrowed down to forty-four clones with preliminarily favorable binding characteristics; among these hybridoma clones, we have identified seven which preferentially bind to HPV-positive cancer cells. We then identified the binding targets of three clones via immunoprecipitation and mass spectrometry. These targets are integrin alpha6 beta4 (ITGA6, ITGB4), tissue factor (F3) and keratin 8 (KRT8) respectively. The bioinformatics-based approach identified several surface proteins that are differentially overexpressed in HPV-positive HNSCC and at levels significantly higher than found in normal control tissue. Evaluation of gene and protein expression in cancer cell lines and/or patient tissue validated several genes identified by the deconvolution approach, including: ROR2, a non-canonical WNT member not previously associated with head and neck cancer; and LY6K, a cancer-testis antigen that overexpressed in both HPV-positive and negative HNSCC. We propose targeting membrane-expressed antigens on HPV-related cancer cells as a platform for further development of novel tumor imaging and therapeutic approaches for HNSCC and other HPV-associated cancers. Citation Format: Hsuan-Chen Liu, Ivenise Carrero, Falguni Parikh, Thomas Kraus, Thomas M. Moran, Mathew J. Ellis, Elizabeth Y. Chiao, Aleksandar Milosavljevic, Andrew G. Sikora. Concurrent in vitro and in silico approach for the identification of surface proteome targets on HPV-associated cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2124.

Plasma membrane proteins mainly function to transmit external signals into the cell. Many plasma membrane receptor tyrosine kinases (e.g., HER2 and EGFR) are known to mediate oncogenic progression, making them prime targets for cancer therapy. Recently, it has become important to identify plasma membrane proteins that are differentially expressed in normal versus cancer cells, in drug-sensitive versus drug-resistant cells, or among tumor cells that metastasize to different organ sites because these differentially expressed membrane proteins may lead to the identification of therapeutic targets or diagnostic markers. In addition, there is an increased interest in identifying cell-surface proteins that could serve as markers for stem cells, progenitor cells, or cells of different lineages. Traditionally, membrane isolation requires multiple centrifugation steps to isolate different organelles based on their density. With the advent of affinity matrix technology, it is possible to separate organelles based on their molecular differences. A defining characteristic of the plasma membrane is that plasma membrane proteins are more extensively glycosylated than are intracellular membrane proteins. As a result, affinity chromatography employing lectin, a carbohydrate-binding protein, is commonly used to isolate plasma membrane proteins. We have extended this concept for plasma membrane isolation by using concanavalin A (ConA), a lectin with mannose specificity. Here we describe a protocol that uses immobilized ConA bound to magnetic beads to isolate plasma membranes from homogenized cell lysates. The captured plasma membrane proteins are then solubilized from the ConA-magnetic beads by detergents in the presence of a competing sugar, methyl α-mannopyranoside.

HPV-driven head and neck squamous cell carcinoma (HNSCC) are among the fastest growing cancers. Although, HPV-HNSCC patients have an overall favorable prognosis, a significant number of patients relapse post-treatment, and currently there is no specific therapeutic approach targeting the unique biology of HPV-driven HNSCC. Our ongoing efforts to target HPV-HNSCC focus on the identification of cell surface antigens that are upregulated by HPV infection. We have developed an "antigen-agnostic" approach for generating HPV-HNSCC-targeting monoclonal antibodies for cancer diagnosis and treatment that does not rely on prior identification of target antigens. We used HPV-HNSCC membrane fractions to immunize recipient mice and generated HPV-specific hybridomas. We then screened five thousand hybridoma colonies by flow cytometry to test the specificity of binding to HPV-positive cancer cell lines (2HNSCC and 2 Cervical Cancer) and HPV-negative cancer cell lines (4 HNSCC and 1 CC). After primary screening, we narrowed down to forty-four clones; among these hybridoma clones, 6D8 and 6B3 bound preferentially to HPV-positive cancer cell lines. The binding targets of 6D8 and 6B3 were identified by immunoprecipitation and mass spectrometry; their targets are integrin alpha6 (ITGA6) and tissue factor (F3) respectively. Future work will validate the biological function of these mAbs in in vitro and in vivo models, and continue identifying more binding target of mAbs. We propose mAbs specifically targeting membrane-expressed antigens on HPV-related cancer cells as a potential approach for early diagnosis and targeted therapy. Citation Format: Hsuan-Chen Liu, Falguni Parikh, Thomas Kraus, Thomas Moran, Andrew Sikora. Characterization of monoclonal antibodies specific for HPV-positive head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2211. doi:10.1158/1538-7445.AM2017-2211

Loss of E-cadherin and up-regulation of mesenchymal cadherins, a hallmark of the epithelial–mesenchymal transition, contributes to migration and dissemination of cancer cells. Expression of human cadherin-11 (Cad11), also known as osteoblast cadherin, in prostate cancer increases the migration of prostate cancer cells. How Cad11 mediates cell migration is unknown. Using the human Cad11 cytoplasmic domain in pulldown assays, we identified human angiomotin (Amot), known to be involved in cell polarity, migration, and Hippo pathway, as a component of the Cad11 protein complex. Deletion analysis showed that the last C-terminal 10 amino acids in Cad11 cytoplasmic domain are required for Amot binding. Further, Cad11 preferentially interacts with Amot-p80 than Amot-p130 isoform and binds directly to the middle domain of Amot-p80. Cad11-Amot interaction affects Cad11-mediated cell migration, but not homophilic adhesion, as deletion of Amot binding motif of Cad11 (Cad11-ΔAmot) did not abolish Cad11-mediated cell–cell adhesion of mouse L cells, but significantly reduced Cad11-mediated cell migration of human C4-2B4 and PC3-mm2 prostate cancer cells and human HEK293T cells. Together, our studies identified Amot-p80 as a novel component of the Cad11 complex and demonstrated that Amot-p80 is critical for Cad11-mediated cell migration.—Ortiz, A., Lee, Y.-C., Yu, G., Liu, H.-C., Lin, S.-C., Bilen, M. A., Cho, H., Yu-Lee, L.-Y., Lin, S.-H. Angiomotin is a novel component of cadherin-11/β-catenin/p120 complex and is critical for cadherin-11-mediated cell migration.

A single-layer of breath figure pattern was explored via the dynamical optical images and the temperature evolution. The pattern was prepared with the solution of carbon disulfide (CS(2)) dissolved 1% weight concentration of polystyrene. The evaporation of CS(2) was considered to be the most important role to the formation of the breath figure pattern. The understanding of the breath figures pattern will promote the technique to fabricating an imprinted template with demanded hexagonal structures.

Table S1. Flow cytometry immune microenvironment staining panels. (DOCX 19 kb)

Table S1. Flow cytometry immune microenvironment staining panels. (DOCX 19 kb)

Figure S8. tdLN immune microenvironment data at day 23. Flow cytometry assessment of tdLN was performed at day 23 for all treatment groups and major immune cell subset percentages (among CD45+ cells) are shown. (A) Percentage of CD4+ and CD8+ T cell subsets. (B) Percentage E7 tetramer+ CD8+ T cells. (C) Percentage of Tregs. (D) Percentage of major myeloid subsets. (For A-D, Tukey’s multiple comparison test; N = 2; 7–13 per group). (E) Aggregate flow cytometry scatter plots of CD8+ T cells showing E7 tetramer staining (N = 1, representative of 2; n = 4 aggregate samples per group). *p

Figure S10. CD8+ T cell phenotype microenvironment heatmaps. Flow cytometry assessment of CD8+ T cell phenotype changes at day 23 for all treatment groups, and further assessed at day 33 and 37 for CPR treatment and compared to tumor size-matched control mice. Data is represented as a z-score of the phenotypic marker’s median fluorescence intensity (MFI) among CD8+ T cells represented as z-scores. (A) Tumor and (B) tdLN CD8+ T cell phenotypic marker expression between all treatment groups at day 23 of treatment (N = 2, n = 7–13 per group). (C) tdLN CD8+ T cell phenotypic marker expression at days 23, 33, and 37 of CPR treatment progression compared to size-matched control tumors (N = 2; n = 7–13 per group). (PDF 3118 kb)

Figure S7. Tumor immune microenvironment data time course. Flow cytometry assessment of tumor was performed at day 23, day 33, and day 37 for the CPR treatment group and major immune cell subset percentages (among CD45+ cells) are shown. (A) Percentage of CD4+ and CD8+ T cell subsets. (B) Percentage E7 tetramer+ CD8+ T cells. (C) Percentage of Tregs. (D) Percentage of major myeloid subsets. (For A-D, Tukey’s multiple comparison test; N = 2; 8–13 per group). (E) Aggregate flow cytometry scatter plots of CD8+ T cells showing E7 tetramer staining (N = 1, representative of 2; n = 4 aggregate samples per group). **p

Figure S6. Tumor immune microenvironment data at day 23. Flow cytometry assessment of tumor was performed at day 23 for all treatment groups and major immune cell subset percentages (among CD45+ cells) are shown. (A) Percentage of CD4+ and CD8+ T cell subsets. (B) Percentage E7 tetramer+ CD8+ T cells. (C) Percentage of Tregs. (D) Percentage of major myeloid subsets. (For A-D, Tukey’s multiple comparison test; N = 2; 8–13 per group). (E) Aggregate flow cytometry scatter plots of CD8+ T cells showing E7 tetramer staining (N = 1, representative of 2; n = 4 aggregate samples per group). *p

Figure S5. CPR increases intratumoral M1-like macrophages. Aggregate flow cytometry scatterplots showing MHCII and iNOS expression among tumor-dwelling macrophages at day 23 of treatment (percentages show mean +/− SD; N = 1 representative of 2; n = 4 aggregate samples per group). (PDF 1299 kb)

Figure S4. CTX/L-NIL improves anti-tumor effect of αPD-1/αCTLA-4 and radiation in the B16 syngeneic melanoma tumor model. Subcutaneous established B16-F0 melanoma tumors (day 4 post tumor cell injection) were treated with αPD-1/αCTLA-4 and radiation alone, or combined with CTX/L-NIL immunomodulation (CPR regimen), mice were euthanized when tumors reached 225 mm2. (A) Average tumor area statistically compared at time of first control mouse euthanization (Tukey’s multiple comparison test; N = 1 representative of 2; n = 7–8 per group). (B) Kaplan Meier survival curves with comparison between treatment groups (Log-rank test; N = 2; n = 10–11 per group). *p

Figure S3. CPR regimen induces minimal weight loss and no gross treatment related toxicities. (A) Normalized weight for treated mice over the course of treatment, normalized to mouse weight 1 week after tumor cell inoculation (N = 1 representative of 2; n = 5–9). (B) Image of mouse treated with full CPR regimen approximately 100 days after tumor clearance with white fur visible in location of tumor clearance. (PDF 1600 kb)

Figure S13: Multiplex raw cellular densities. Raw cellular densities from multiplex analysis used for generation of Fig. 4b and c. For all samples N = 1 and n = 15 images per group (3 mice per group with 5 images assessed per tumor). (PDF 1479 kb)

Figure S7. Tumor immune microenvironment data time course. Flow cytometry assessment of tumor was performed at day 23, day 33, and day 37 for the CPR treatment group and major immune cell subset percentages (among CD45+ cells) are shown. (A) Percentage of CD4+ and CD8+ T cell subsets. (B) Percentage E7 tetramer+ CD8+ T cells. (C) Percentage of Tregs. (D) Percentage of major myeloid subsets. (For A-D, Tukey’s multiple comparison test; N = 2; 8–13 per group). (E) Aggregate flow cytometry scatter plots of CD8+ T cells showing E7 tetramer staining (N = 1, representative of 2; n = 4 aggregate samples per group). **p

Figure S11. Myeloid flow cytometry gating strategy. Flow cytometry strategy used for gating myeloid subsets, with labeled cell type above the respective gate. (PDF 2002 kb)

Figure S6. Tumor immune microenvironment data at day 23. Flow cytometry assessment of tumor was performed at day 23 for all treatment groups and major immune cell subset percentages (among CD45+ cells) are shown. (A) Percentage of CD4+ and CD8+ T cell subsets. (B) Percentage E7 tetramer+ CD8+ T cells. (C) Percentage of Tregs. (D) Percentage of major myeloid subsets. (For A-D, Tukey’s multiple comparison test; N = 2; 8–13 per group). (E) Aggregate flow cytometry scatter plots of CD8+ T cells showing E7 tetramer staining (N = 1, representative of 2; n = 4 aggregate samples per group). *p

Figure S1. Immune checkpoint inhibitors alone provide minimal treatment benefit or microenvironment improvement. (A-C) Subcutaneous established mEER tumors (day 17–18 post tumor cell injection) were treated with 6 total doses of αPD-1 (250 μg/dose) and/or αCTLA-4 (100 μg/dose) according to the schedule in (Fig. 1b), mice were euthanized when tumors reached 225 mm2. (A) Kaplan Meier survival curves with comparison between treatment groups (Log-rank test; N = 1 representative of 2; n = 5–12). (B) Flow cytometry immune profiling of treated tumors at 1 week of treatment (day 23) shown as z-scores for major lymphoid and myeloid subtypes between groups (N = 2; n = 9–12 per group). (C) Percentage of major lymphoid subsets (among CD45+ cells) in tdLN at day 23 of treatment for various treatment groups (Tukey’s multiple comparison test; N = 2; n = 9–12 per group). *p

Figure S9. tdLN immune microenvironment data time course. Flow cytometry assessment of tdLN was performed at day 23, day 33, and day 37 for the CPR treatment group and major immune cell subset percentages (among CD45+ cells) are shown. (A) Percentage of CD4+ and CD8+ T cell subsets. (B) Percentage E7 tetramer+ CD8+ T cells. (C) Percentage of Tregs. (D) Percentage of major myeloid subsets. (For A-D, Tukey’s multiple comparison test; N = 2; 7–13 per group). (E) Aggregate flow cytometry scatter plots of CD8+ T cells showing E7 tetramer staining (N = 1, representative of 2; n = 4 aggregate samples per group). *p

Figure S1. Immune checkpoint inhibitors alone provide minimal treatment benefit or microenvironment improvement. (A-C) Subcutaneous established mEER tumors (day 17–18 post tumor cell injection) were treated with 6 total doses of αPD-1 (250 μg/dose) and/or αCTLA-4 (100 μg/dose) according to the schedule in (Fig. 1b), mice were euthanized when tumors reached 225 mm2. (A) Kaplan Meier survival curves with comparison between treatment groups (Log-rank test; N = 1 representative of 2; n = 5–12). (B) Flow cytometry immune profiling of treated tumors at 1 week of treatment (day 23) shown as z-scores for major lymphoid and myeloid subtypes between groups (N = 2; n = 9–12 per group). (C) Percentage of major lymphoid subsets (among CD45+ cells) in tdLN at day 23 of treatment for various treatment groups (Tukey’s multiple comparison test; N = 2; n = 9–12 per group). *p