Compartmentalized spatial profiling of the tumor microenvironment in head and neck squamous cell carcinoma identifies immune checkpoint molecules and tumor necrosis factor receptor superfamily members as biomarkers of response to immunotherapy.

Mucosal head and neck squamous cell carcinoma (HNSCC) are the seventh most common cancer, with approximately 50% of patients living beyond 5 years. Immune checkpoint inhibitors (ICIs) have shown promising results in patients with recurrent or metastatic (R/M) disease, however, only a subset of patients benefit from immunotherapy. Studies have implicated the tumor microenvironment (TME) of HNSCC as a major factor in therapy response, highlighting the need to better understand the TME, particularly by spatially resolved means to determine cellular and molecular components. Here, we employed targeted spatial profiling of proteins on a cohort of pre-treatment tissues from patients with R/M disease to identify novel biomarkers of response within the tumor and stromal margins. By grouping patient outcome categories into response or non-response, based on Response Evaluation Criteria in Solid Tumors (RECIST) we show that immune checkpoint molecules, including PD-L1, B7-H3, and VISTA, were differentially expressed. Patient responders possessed significantly higher tumor expression of PD-L1 and B7-H3, but lower expression of VISTA. Analysis of response subgroups indicated that tumor necrosis factor receptor (TNFR) superfamily members including OX40L, CD27, 4-1BB, CD40, and CD95/Fas, were associated with immunotherapy outcome. CD40 expression was higher in patient-responders than non responders, while CD95/Fas expression was lower in patients with partial response (PR) relative to those with stable disease (SD) and progressive disease (PD). Furthermore, we found that high 4-1BB expression in the tumor compartment, but not in the stroma, was associated with better overall survival (OS) (HR= 0.28, p-adjusted= 0.040). Moreover, high CD40 expression in tumor regions (HR= 0.27, p-adjusted= 0.035), and high CD27 expression in the stroma (HR= 0.2, p-adjusted=0.032) were associated with better survival outcomes. Taken together, this study supports the role of immune checkpoint molecules and implicates the TNFR superfamily as key players in immunotherapy response in our cohort of HNSCC. Validation of these findings in a prospective study is required to determine the robustness of these tissue signatures.

NEO212, a conjugate of temozolomide and perillyl alcohol, blocks the endothelial-to-mesenchymal transition in tumor-associated brain endothelial cells in glioblastoma.

As the endothelial-to-mesenchymal transition (EndMT) supports the pro-angiogenic and invasive characteristics of glioblastoma multiforme (GBM), blocking this process would be a promising approach to inhibit tumor progression and recurrence. Here, we demonstrate that glioma stem cells (GSC) induce EndMT in brain endothelial cells (BEC). TGF-ß signaling is necessary, but not sufficient to induce this EndMT process. Cell-to-cell contact and the contribution of Notch signaling are also required. NEO212, a conjugate of temozolomide and perillyl alcohol, blocks EndMT induction and reverts the mesenchymal phenotype of tumor-associated BEC (TuBEC) by blocking TGF-ß and Notch pathways. Consequently, NEO212 reduces the invasiveness and pro-angiogenic properties associated with TuBEC, without affecting control BEC. Intracranial co-implantation of BEC and GSC in athymic mice showed that EndMT occurs in vivo, and can be blocked by NEO212, supporting the potential clinical value of NEO212 for the treatment of GBM.

Monoamine oxidase A (MAO A) inhibitors decrease glioma progression.

Glioblastoma (GBM) is an aggressive brain tumor which is currently treated with temozolomide (TMZ). Tumors usually become resistant to TMZ and recur; no effective therapy is then available. Monoamine Oxidase A (MAO A) oxidizes monoamine neurotransmitters resulting in reactive oxygen species which cause cancer. This study shows that MAO A expression is increased in human glioma tissues and cell lines. MAO A inhibitors, clorgyline or the near-infrared-dye MHI-148 conjugated to clorgyline (NMI), were cytotoxic for glioma and decreased invasion in vitro. Using the intracranial TMZ-resistant glioma model, clorgyline or NMI alone or in combination with low-dose TMZ reduced tumor growth and increased animal survival. NMI was localized specifically to the tumor. Immunocytochemistry studies showed that the MAO A inhibitor reduced proliferation, microvessel density and invasion, and increased macrophage infiltration. In conclusion, we have identified MAO A inhibitors as potential novel stand-alone drugs or as combination therapy with low dose TMZ for drug-resistant gliomas. NMI can also be used as a non-invasive imaging tool. Thus has a dual function for both therapy and diagnosis.

A novel drug conjugate, NEO212, targeting proneural and mesenchymal subtypes of patient-derived glioma cancer stem cells.

Glioblastoma multiforme (GBM), a highly malignant brain tumor, accounts for half of all gliomas. Despite surgery, radiation and chemotherapy, the median survival is between 12 and 15 months. The poor prognosis is due to tumor recurrence attributed to chemoresistant glioma cancer stem cells (GSCs). Here we examined the effects of a novel compound NEO212, which is composed of two covalently conjugated anti-cancer compounds - temozolomide (TMZ) and perillyl alcohol (POH), on GSCs expressing either the proneural or mesenchymal gene signatures. These GSCs were obtained from patient-derived tumor tissue. Our findings demonstrate that NEO212 is 10 fold more cytotoxic to GSCs than TMZ (standard-of-care). Furthermore, NEO212 is effective against both proneural and clinically aggressive mesenchymal GSC subtypes. The mechanism of NEO212 mediated-cytotoxicity is through double-strand DNA breaks and apoptosis. In vivo studies show that NEO212 significantly delays tumor growth of both proneural and mesenchymal tumor stem cell populations. Patient-derived GSCs and tumors derived from these cells are highly reflective of the heterogeneity in human GBM. The efficacy of NEO212 against both GSC subtypes indicates that NEO212 has great clinical potential to effectively target GBM.

Tumor vasculature and glioma stem cells: Contributions to glioma progression.

Glioblastoma multiforme (GBM), the most malignant of brain tumors, is characterized by extensive vascularization and a high degree of invasion. The current standard of care is not very effective, resulting in tumor recurrence with patients rarely surviving over 2 years. This tumor recurrence is attributed to the presence of chemo and radiation resistant glioma stem cells (GSCs). These cells are associated with vascular niches which regulate GSC self-renewal and survival. Recent studies suggest that while blood vessels support glioma stem cells, these tumor cells in turn may regulate and contribute to the tumor vasculature by transdifferentiating into endothelial cells directly or through the secretion of regulatory growth factors such as vascular endothelial growth factor (VEGF) and hepatoma derived growth factor (HDGF). The relationship between the tumor vasculature and the glioma stem cells is the subject of this review.

Effects of convection-enhanced delivery of bevacizumab on survival of glioma-bearing animals.

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

A novel temozolomide analog, NEO212, with enhanced activity against MGMT-positive melanoma in vitro and in vivo.

The alkylating agent temozolomide (TMZ) represents an important component of current melanoma therapy, but overexpression of O6-methyl-guanine DNA methyltransferase (MGMT) in tumor cells confers resistance to TMZ and impairs therapeutic outcome. We investigated a novel perillyl alcohol (POH)-conjugated analog of TMZ, NEO212, for its ability to exert anticancer activity against MGMT-positive melanoma cells. Human melanoma cells with variable MGMT expression levels were treated with NEO212, TMZ, or perillyl alcohol in vitro and in vivo, and markers of DNA damage and apoptosis, and tumor cell growth were investigated. NEO212 displayed substantially greater anticancer activity than any of the other treatments. It reduced colony formation of MGMT-positive cells up to eight times more effectively than TMZ, and much more potently induced DNA damage and cell death. In a nude mouse tumor model, NEO212 showed significant activity against MGMT-positive melanoma, whereas TMZ, or a mix of TMZ plus POH, was ineffective. At the same time, NEO212 was well tolerated. NEO212 may have potential as a more effective therapy for advanced melanoma, and should become particularly suitable for the treatment of patients with MGMT-positive tumors.

Sequential administration of carbon nanotubes and near-infrared radiation for the treatment of gliomas.

The objective was to use carbon nanotubes (CNT) coupled with near-infrared radiation (NIR) to induce hyperthermia as a novel non-ionizing radiation treatment for primary brain tumors, glioblastoma multiforme (GBM). In this study, we report the therapeutic potential of hyperthermia-induced thermal ablation using the sequential administration of carbon nanotubes (CNT) and NIR. In vitro studies were performed using glioma tumor cell lines (U251, U87, LN229, T98G). Glioma cells were incubated with CNTs for 24 h followed by exposure to NIR for 10 min. Glioma cells preferentially internalized CNTs, which upon NIR exposure, generated heat, causing necrotic cell death. There were minimal effects to normal cells, which correlate to their minimal uptake of CNTs. Furthermore, this protocol caused cell death to glioma cancer stem cells, and drug-resistant as well as drug-sensitive glioma cells. This sequential hyperthermia therapy was effective in vivo in the rodent tumor model resulting in tumor shrinkage and no recurrence after only one treatment. In conclusion, this sequence of selective CNT administration followed by NIR activation provides a new approach to the treatment of glioma, particularly drug-resistant gliomas.

NEO212, temozolomide conjugated to perillyl alcohol, is a novel drug for effective treatment of a broad range of temozolomide-resistant gliomas.

Patients with glioblastoma multiforme (GBM), a malignant primary brain tumor, inevitably develop resistance to standard-of-care chemotherapy, temozolomide. This study explores the effects of the novel agent NEO212, a conjugate of temozolomide to perillyl alcohol, on temozolomide-resistant gliomas. NEO212 was tested for cytotoxic activity on three human temozolomide-resistant glioma cell lines, which were resistant to temozolomide based on overexpression of the base excision repair (BER) pathway, mismatch repair (MMR) deficiency, or overexpression of O(6) methyl-guanine-DNA methyltransferase (MGMT). BER expression was evaluated by Western blotting and PARP activity. MMR deficiency was determined by Western blotting and microsatellite instability. MGMT overexpression was evaluated by Western blotting and O(6)-benzylguanine (O(6)BG) inhibition. For in vivo evaluation of NEO212, temozolomide-resistant glioma cells were implanted into immune-incompetent mice, and NEO212 was administered. NEO212, at equimolar concentrations of temozolomide, was more cytotoxic for temozolomide-resistant cells than temozolomide and not toxic to normal cells. NEO212-induced cell death in temozolomide-resistant glioma cells was independent of such mechanisms of resistance as high levels of MGMT, MMR deficiencies, or overexpression of BER proteins. NEO212 functions as a DNA alkylating agent, similar to temozolomide; however, this novel conjugate is unique for it may induce endoplasmic reticulum (ER) stress and inhibits autophagy. In vivo studies show that NEO212 reduces intracranial tumor growth and increases animal survival without significant toxicity. These results demonstrate that NEO212 is an effective drug against malignant gliomas that can be used for a broad range of newly diagnosed and temozolomide-resistant gliomas.

Perillyl alcohol for the treatment of temozolomide-resistant gliomas.

Perillyl alcohol (POH) is a monoterpene that has been used orally for the treatment of systemic cancer. However, when used orally significant gastrointestinal side effects and lack of overall efficacy were documented. Recently, in a phase II trial in Brazil for the treatment of temozolomide (TMZ)-resistant malignant gliomas, POH was well tolerated when administered intranasally. The present study explores the effects and mechanisms of POH on TMZ-sensitive and TMZ-resistant glioma cells. In vitro studies showed that POH was cytotoxic to TMZ-resistant as well as TMZ-sensitive glioma cells, and this effect was independent of O(6)-methylguanine-DNA methyltransferase expression. POH induced cytotoxicity, in part, through the endoplasmic reticulum (ER) stress pathway as shown by the increased expression of glucose-regulated protein-78 (GRP78), activating transcription factor 3, and C/EBP-homologous protein. In addition, POH impeded survival pathways, such as mTOR and Ras. As well, POH reduced the invasive capacity of sensitive and resistant glioma cells. POH alone and/or in combination with other ER stress-inducing cytotoxic drugs (i.e., 2, 5-dimethyl-celecoxib, nelfinavir) further induced apoptosis in TMZ-sensitive and TMZ-resistant glioma cells. To show whether intranasal delivery of POH was effective for the treatment of TMZ-resistant gliomas, animals bearing intracranial tumors were given POH intranasally. Animals treated through intranasal administration of POH exhibited a decrease in tumor growth and an increase in survival. Our data show that POH is an effective anti-glioma cytotoxic agent for TMZ-resistant gliomas when administered intranasally.

Noscapine inhibits tumor growth in TMZ-resistant gliomas.

Noscapine, a common oral antitussive agent, has been shown to have potent antitumor activity in a variety of cancers. Treatment of glioblastoma multiforme (GBM) with temozolomide (TMZ), its current standard of care, is problematic because the tumor generally recurs and is then resistant to this drug. We therefore investigated the effects of noscapine on human TMZ-resistant GBM tumors. We found that noscapine significantly decreased TMZ-resistant glioma cell growth and invasion. Using the intracranial xenograft model, we showed that noscapine increased survival of animals with TMZ-resistant gliomas. Thus noscapine can provide an alternative therapeutic approach for the treatment of TMZ-resistant gliomas.

A critical role for GRP78/BiP in the tumor microenvironment for neovascularization during tumor growth and metastasis.

Glucose-regulated protein 78 (GRP78)/BiP is a multifunctional protein which plays a major role in endoplasmic reticulum (ER) protein processing, protein quality control, maintaining ER homeostasis, and controlling cell signaling and viability. Previously, using a transgene-induced mammary tumor model, we showed that Grp78 heterozygosity impeded cancer growth through suppression of tumor cell proliferation and promotion of apoptosis and the Grp78(+/-) mice exhibited dramatic reduction (70%) in the microvessel density (MVD) of the endogenous mammary tumors, while having no effect on the MVD of normal organs. This observation suggests that GRP78 may critically regulate the function of the host vasculature within the tumor microenvironment. In this article, we interrogated the role of GRP78 in the tumor microenvironment. In mouse tumor models in which wild-type (WT), syngeneic mammary tumor cells were injected into the host, we showed that Grp78(+/-) mice suppressed tumor growth and angiogenesis during the early phase but not during the late phase of tumor growth. Growth of metastatic lesions of WT, syngeneic melanoma cells in the Grp78(+/-) mice was potently suppressed. We created conditional heterozygous knockout of GRP78 in the host endothelial cells and showed severe reduction of tumor angiogenesis and metastatic growth, with minimal effect on normal tissue MVD. Furthermore, knockdown of GRP78 expression in immortalized human endothelial cells showed that GRP78 is a critical mediator of angiogenesis by regulating cell proliferation, survival, and migration. Our findings suggest that concomitant use of current chemotherapeutic agents and novel therapies against GRP78 may offer a powerful dual approach to arrest cancer initiation, progression, and metastasis.