Vascular attack by 5,6-dimethylxanthenone-4-acetic acid combined with B7.1 (CD80)-mediated immunotherapy overcomes immune resistance and leads to the eradication of large tumors and multiple tumor foci

The promise of cancer immunotherapy is that it will not only eradicate primary tumors but will generate systemic antitumor immunity capable of destroying distant metastases. A major problem that must first be surmounted relates to the immune resistance of large tumors. Here we reveal that immune resistance can be overcome by combining immunotherapy with a concerted attack on the tumor vasculature. The functionally related antitumor drugs 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and flavone acetic acid (FAA), which cause tumor vasculature collapse and tumor necrosis, were used to attack the tumor vasculature, whereas the T-cell costimulator B7.1 (CD80), which costimulates T-cell proliferation via the CD28 pathway, was used to stimulate antitumor immunity. The injection of cDNA (60–180 µg) encoding B7.1 into large EL-4 tumors (0.8 cm in diameter) established in C57BL/6 mice, followed 24 h later by i.p. administration of either DMXAA (25 mg/kg) or FAA (300 mg/kg), resulted in complete tumor eradication within 2–6 weeks. In contrast, monotherapies were ineffective. Both vascular attack and B7.1 immunotherapy led to up-regulation of heat shock protein 70 on stressed and dying tumor cells, potentially augmenting immunotherapy. Remarkably, large tumors took on the appearance of a wound that rapidly ameliorated, leaving perfectly healed skin. Combined therapy was mediated by CD8+ T cells and natural killer cells, accompanied by heightened and prolonged antitumor cytolytic activity (P < 0.001), and by a marked increase in tumor cell apoptosis. Cured animals completely rejected a challenge of 1 x 107 parental EL-4 tumor cells but not a challenge of 1 x 104 Lewis lung carcinoma cells, demonstrating that antitumor immunity was tumor specific. Adoptive transfer of 2 x 108 splenocytes from treated mice into recipients bearing established (0.8 cm in diameter) tumors resulted in rapid and complete tumor rejection within 3 weeks. Although DMXAA and B7.1 monotherapies are complicated by a narrow range of effective doses, combined therapy was less dosage dependent. Thus, a broad range of amounts of B7.1 cDNA were effective in combination with 25 mg/kg DMXAA. In contrast, DMXAA, which has a very narrow range of high active doses, was effective at a low dose (18 mg/kg) when administered with a large amount (180 µg) of B7.1 cDNA. Importantly, combinational therapy generated heightened antitumor immunity, such that gene transfer of B7.1 into one tumor, followed by systemic DMXAA treatment, led to the complete rejection of multiple untreated tumor nodules established in the opposing flank. These findings have important implications for the future direction and utility of cancer immunotherapies aimed at harnessing patients’ immune responses to their own tumors.

The PKCÁ-D294G mutant found in pituitary and thyroid tumors fails to transduce extracellular signals

Protein kinase C (PKC) is a key regulator of cell proliferation, differentiation, and apoptosis and is one of the drug targets of anticancer therapy. Recently, a single point mutation (D294G) in PKCα has been found in pituitary and thyroid tumors with more invasive phenotype. Although the PKCα-D294G mutant is implicated in the progression of endocrine tumors, no apparent biochemical/cell biological abnormalities underlying tumorigenesis with this mutant have been found. We report here that the PKCα-D294G mutant is unable to bind to cellular membranes tightly despite the fact that it translocates to the membrane as efficiently as the wild-type PKCα upon treatment of phorbol ester. The impaired membrane binding is associated with this mutant's inability to transduce several antitumorigenic signals as it fails to mediate phorbol ester–stimulated translocation of myristoylated alanine–rich protein kinase C substrate (MARCKS), to activate mitogen-activated protein kinase and to augment melatonin-stimulated neurite outgrowth. Thus, the PKCα-D294G is a loss-of-function mutation. We propose that the wild-type PKCα may play important antitumorigenic roles in the progression of endocrine tumors. Therefore, developing selective activators instead of inhibitors of PKCα might provide effective pharmacological interventions for the treatment of certain endocrine tumors.

Low-dose metronomic paclitaxel chemotherapy suppresses breast tumors and metastases in mice

This study investigated the use of low-dose metronomic (LDM) chemotherapy with paclitaxel in a highly metastatic mouse model of 4T1 breast cancers, and compared it with the maximum tolerable dose (MTD) therapy. LDM therapy displayed a stronger anti-tumor activity in suppressing primary and metastatic breast tumors with less degree of side effects, and stronger anti-angiogenic and anti-lymphangiogenic activities than MTD therapy. But MTD therapy showed stronger pro-apoptotic and anti-proliferative activities in situ. Paclitaxel therapy downregulated expression of vascular endothelial growth factor receptor-2 (VEGFR2) and up-regulated expression of thrombospondin-1. The results support the application of paclitaxel LDM therapy to treat advanced breast cancer.

Effects of survivin antagonists of growth of established tumors and B7-1 immunogene therapy

Background: Survivin, a member of the inhibitor of apoptosis (IAP) protein family, is detectable in most types of cancer, and its presence is associated with a poor prognosis. We determined the effects of gene-based therapies that inhibit survivin function in a mouse tumor model. Methods: Using five to six mice per treatment group, we injected tumors derived from mouse EL-4 thymic lymphoma cells with plasmids encoding antisense survivin, a dominant-negative mutant survivin, and the T-cell costimulator B7-1. Expression of endogenous survivin and the proteins encoded by the injected plasmids were examined by immunohistochemical staining of tumor sections and by western blot and flow cytometry analyses of isolated tumor cells. Tumor growth, the generation of antitumor cytotoxic T-lymphocyte (CTL) activity, apoptosis, and the contribution of leukocyte subsets to antitumor activity were measured. All statistical tests were two-sided. Results: Large (1.0-cm diameter) tumors had approximately 10-fold more survivin than small (0.2-cm diameter) tumors. At 28 days after injection, antisense and dominant-negative mutant survivin plasmids statistically significantly inhibited the growth of both small (P = .006 and P = .0018, respectively) and large (P<.001 for both plasmids) EL-4 tumors compared with tumors injected with empty plasmid. The growth of large tumors was further inhibited by intratumoral injection with antisense survivin and B7-1 (P = .004); thus, inhibition of survivin expression renders large tumors susceptible to B7-1-mediated immunotherapy. Mice whose tumors were completely eradicated by injection of B7-1 remained tumor free for 26 days after re-injection with EL-4 cells (when the experiment ended). Compared with tumors injected with empty plasmid, tumors injected with survivin-based plasmids had increased apoptosis, and animals bearing such tumors generated more antitumor CTLs. Conclusion: Intratumoral injection of plasmids that block survivin expression and stimulate the generation of tumor-specific CTLs may be beneficial for the treatment of large lymphomas.

Regression of solid tumors by engineered overexpression of von Hippel-Lindau tumor suppressor protein and antisense hypoxia-inducible factor-1alpha

The von Hippel-Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the alpha subunits of hypoxia-inducible factors (HIFs) responsible for stimulating tumor angiogenesis and glycolysis, targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to the development of sporadic renal cell carcinomas (RCCs). In the present study, we sought to determine whether engineered overexpression of pVHL in tumors other than RCC can inhibit tumor growth, either as a monotherapy, or in combination with antisense HIF-1alpha therapy. Intratumoral injection of subcutaneous EL-4 thymic lymphomas with an expression plasmid encoding pVHL resulted in the downregulation of HIF-1alpha and vascular endothelial growth factor (VEGF). There was a concomitant reduction in tumor angiogenesis and increased tumor cell apoptosis due in part to downregulation of Bcl-2 expression. VHL therapy resulted in the complete regression of small (0.1 cm diameter) tumors whereas, in contrast, large (0.4 cm diameter) EL-4 tumors were only slowed in their growth. Nevertheless, large tumors completely regressed in response to intratumoral injection of a combination of antisense HIF-1alpha and VHL plasmids. Combination therapy resulted in increased losses of HIF-1alpha, VEGF, and tumor blood vessels, and increased tumor cell apoptosis. These novel results suggest that synergistic therapies that simultaneously block the expression or function of HIF-1alpha, and enhance the expression or function of VHL may be beneficial in the treatment of cancer.

Goblet cell carcinoid tumors (adenocarcinoid) of the appendix

Purpose: Goblet cell appendiceal carcinoids represent rare tumors that exhibit histologic features of both adenocarcinomas and neuroendocrine tumors. We present the long-term results of a series of 15 patients, focusing on clinical manifestations, diagnosis, and management.

Methods: Eight male and seven female patients (median age, 52.8 years) were included. Final diagnosis was confirmed by histology. Patients were evaluated clinically, biochemically, and radiologically every four months. Median follow-up was 30 months.

Results: The majority of patients (7/15) presented with symptoms compatible with acute appendicitis. Right hemicolectomy was performed in all except one, who subsequently developed metastases. Three patients had metastases at previous diagnosis. Plasma chromogranin-A was slightly elevated in two of them, while urinary 5-hydroxy-indol-acetic acid was normal. 111Indium-labeled octreotide scintigraphy was positive only in two of the four patients with metastases. Ki67 index was greater than 20 percent in all of them, while in only one with local tumor. Combination chemotherapy with either cisplatin plus etoposide or with 5-fluorouracil, cisplatin, and streptozotocin was administered to all patients with metastases resulting in temporary stabilization of disease. Twelve patients are alive, while three died of their disease 9, 13, and 14 months after diagnosis.

Conclusions: The diagnostic value of chromogranin-A, urinary 5-hydroxy-indol-acetic acid, and 111Indium-labeled octreotide scintigraphy seems to be limited in these tumors. Ki67 index appears to predict tumor behavior. Right hemicolectomy may reduce the risk of developing metastases. Chemotherapy may have efficacy in metastatic disease, however, more data are required to determine this and the optimal regimen.

Thrombocytopenia in solid tumors

Thrombocytopenia in patients with solid malignancy can be caused by bone marrow involvement or toxicity from anticancer therapy; however, it could rarely be the first presentation of a tumor such as breast cancer or lymphoma. Hematological paraneoplastic syndromes such as paraneoplastic thrombocytopenia and/or immune thrombocytopenic purpura (ITP) are well described as secondary findings simultaneously with malignancies such as breast cancer and lymphoma. Other hematological conditions such as severe amegakaryocytic thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), and myelodysplastic syndromes (MDS) have also rarely been described as a possible paraneoplastic process complicating solid tumors. On the one hand, occult disseminated malignancy may mimic ITP and TTP, leading to diagnostic and therapeutic problems. On the other hand, thrombocytopenia could be the first manifestation of cancer.

Management of advanced neuroendocrine tumors with hepatic metastasis

Neuroendocrine tumors (NETs) in general and specifically these gastroenteropancreatic-neuroendocrine tumors often present a considerable diagnostic and therapeutic challenge, especially those that behave in an aggressive fashion. The majority of tumors are diagnosed at a stage that the only curative treatment, radical surgical intervention, is no longer an option and thus long-term therapy with somatostatin analogs is focused on symptom amelioration and in the improvement of quality of life. Although biotherapy is currently the most efficient treatment to achieve palliation, conventional chemotherapy may have some utility in undifferentiated or highly proliferating neuroendocrine carcinomas and pancreatic NETs. Hepatic metastases, depending on size, location, and number may be amenable to surgical resection or radiofrequency ablation. If surgery is not feasible, embolization either alone (bland), in combination with chemotherapeutic agents, or using radioactive microspheres can be used. Peptide receptor targeted radiotherapy using radiolabeled octapeptide analogs (⁹⁰Yttrium or ¹⁷⁷Lutetium-octreotide) may lead to reduction in tumor size, but in most circumstances has a tumor stabilizing effect. A variety of antiangiogenesis and growth factor-targeted agents have been evaluated, but to date, the results have failed to meet our expectations.

Frequency of excisions and yields of malignant skin tumors in a population-based screening intervention of 360 288 whole-body examinations

Objective To explore the frequency of excisions and yields of histopathologically confirmed skin cancer.

Design A population-based skin cancer screening intervention (the SCREEN project) in the German state of Schleswig-Holstein (July 1, 2003, to June 30, 2004).

Setting Physician offices. Participants could choose between nondermatologist physicians and dermatologists for their initial whole-body skin examination. All screening physicians received a mandatory 8-hour training course.

Participants Inhabitants of Schleswig-Holstein 20 years or older with statutory health insurance (N = 360 288).

Main Outcome Measures Frequency of excisions and yields of malignant skin tumors (malignant melanomas [MMs], basal cell carcinomas [BCCs], and squamous cell carcinomas [SCCs]), stratified by sex and age.

Results Overall, 15 983 excisions were performed (1 of 23 screenees). A total of 3103 malignant skin tumors were diagnosed in 2911 persons: 585 MMs, 1961 BCCs, 392 SCCs, and 165 other malignant skin tumors. Overall, 116 persons (3103 of 360 288) had to be screened to find 1 malignant tumor, with 1 of 620 for MM, 1 of 184 for BCC, and 1 of 920 for SCC. Twenty excisions were performed to find 1 melanoma in men 65 years and older, but more than 50 excisions were required to find 1 melanoma in men aged between 20 and 49 years.

Conclusions The results of SCREEN suggest a high yield of malignant skin tumors in a large-scale population-based screening project. We found that a high number of excisions was performed in the youngest screenees with an associated low yield, suggesting a need in screener training to emphasize a more conservative attitude toward excisions in young screenees.

Transfection of MDA-MB-231 human breast carcinoma cells with bone sialoprotein (BSP) stimulates migration and invasion in vitro and growth of primary and secondary tumors in nude mice

We have investigated the role of bone sialoprotein (BSP), a secreted glycoprotein normally found in bone, in breast cancer progression. To explore functions for BSP in human breast cancer invasion and metastasis, the full-length BSP cDNA was transfected into the MDA-MB-231-BAG human breast cancer cell line under the control of the CMV promoter. Clones expressing BSP and vector control clones were isolated. BSP producing clones showed increased monolayer wound healing, a faster rate of stellate outgrowth in Matrigel and increased rate of invasion into a collagen matrix when compared to control clones. Clones were also examined in models of breast cancer growth and metastasis in vivo. BSP transfected clones showed an increased rate of primary tumor growth following mammary fat pad injection of nude mice. BSP transfected clones and vector control clones metastasized to soft organs and bone at a similar rate after intra-cardiac injection as determined by real-time PCR and X-ray analysis. Although these organs were targets for both BSP transfected and non-transfected cells, the size of the metastatic lesion was shown to be significantly larger for BSP expressing clones. This was determined by real-time PCR analysis for soft organs and by X-ray analysis of bone lesions. For bone this was confirmed by intra-tibial injections of cells in nude mice. We conclude that BSP acts to drive primary and secondary tumor growth of breast cancers in vivo.

Analysis of 177Lu-octreotate therapy-induced DNA damage in peripheral blood lymphocytes of patients with neuroendocrine tumors

Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) can lead to cell death, genome instability and carcinogenesis. Immunofluorescence detection of phosphorylated histone variant H2AX (γ-H2AX) is a reliable and sensitive technique to monitor external beam IR-induced DSBs in peripheral blood lymphocytes (PBL). Here, we investigated whether γ-H2AX could be used as an in vivo marker to assess normal tissue toxicity after extended internal irradiation with (177)Lu-DOTA-octreotate peptide receptor radionuclide therapy (LuTate PRRT) of neuroendocrine tumors.

Foxp3 expression in macrophages associated with RENCA tumors in mice.

The transcription factor Foxp3 represents the most specific functional marker of CD4+ regulatory T cells (TRegs). However, previous reports have described Foxp3 expression in other cell types including some subsets of macrophages, although there are conflicting reports and Foxp3 expression in cells other than Treg is not well characterized. We performed detailed investigations into Foxp3 expression in macrophages in the normal tissue and tumor settings. We detected Foxp3 protein in macrophages infiltrating mouse renal cancer tumors injected subcutaneously or in the kidney. Expression was demonstrated using flow cytometry and Western blot with two individual monoclonal antibodies. Further analyses confirmed Foxp3 expression in macrophages by RT PCR, and studies using ribonucleic acid-sequencing (RNAseq) demonstrated a previously unknown Foxp3 messenger (m)RNA transcript in tumor-associated macrophages. In addition, depletion of Foxp3+ cells using diphtheria toxin in Foxp3DTR mice reduced the frequency of type-2 macrophages (M2) in kidney tumors. Collectively, these results indicate that tumor-associated macrophages could express Foxp3.

Targeting the sodium iodide symporter (NIS) and apoptotic genes in extrathyroidal tumors.

Sodium Iodide Symporter (NIS), a therapeutic gene, was studied for the first time in retinoblastoma (RB) correlating the expression with clinicopathological invasiveness of the tumor. The specificity of EpCAM based NIS gene therapy was demonstrated in breast cancer cell as a proof of concept model via 1) EpCAM as tissue specific promoter and 2) nanoformulation, both of which showed encouraging outcomes. In addition, for the first time the upregulated expression of splice variants of survivin, Bax and Bcl-2 in RB tumors was explored indicating their possible role in tumor progression through apoptosis dysregulation. Thus, the above study achieved a profound knowledge about NIS and apoptotic genes in extrathyroidal tumors.

Superior performance of aptamer in tumor penetration over antibody : implication of aptamer-based theranostics in solid tumors

Insufficient penetration of therapeutic agents into tumor tissues results in inadequate drug distribution and lower intracellular concentration of drugs, leading to the increase of drug resistance and resultant failure of cancer treatment. Targeted drug delivery to solid tumors followed by complete drug penetration and durable retention will significantly improve clinical outcomes of cancer therapy. Monoclonal antibodies have been commonly used in clinic for cancer treatment, but their limitation of penetrating into tumor tissues still remains because of their large size. Aptamers, as "chemical antibodies", are 15-20 times smaller than antibodies. To explore whether aptamers are superior to antibodies in terms of tumor penetration, we carried out the first comprehensive study to compare the performance of an EpCAM aptamer with an EpCAM antibody in theranostic applications. Penetration and retention were studied in in vitro three-dimensional tumorspheres, in vivo live animal imaging and mouse colorectal cancer xenograft model. We found that the EpCAM aptamer can not only effectively penetrate into the tumorsphere cores but can also be retained by tumor sphere cells for at least 24 h, while limited tumor penetration by EpCAM antibody was observed after 4 h incubation. As observed from in vivo live animal imaging, EpCAM aptamers displayed a maximum tumor uptake at around 10 min followed by a rapid clearance after 80 min, while the signal of peak uptake and disappearance of antibody appeared at 3 h and 6 h after intravenous injection, respectively. The signal of PEGylated EpCAM aptamers in xenograft tumors was sustained for 26 h, which was 4.3-fold longer than that of the EpCAM antibody. Consistently, there were 1.67-fold and 6.6-fold higher accumulation of PEGylated aptamer in xenograft tumors than that of antibody, at 3 h and 24 h after intravenous administration, respectively. In addition, the aptamer achieved at least a 4-time better tumor penetration in xenograft tumors than that of the antibody at a 200 μm distances from the blood vessels 3 h after intravenous injection. Taken together, these data indicate that aptmers are superior to antibodies in cancer theranostics due to their better tumor penetration, more homogeneous distribution and longer retention in tumor sites. Thus, aptamers are promising agents for targeted tumor therapeutics and molecular imaging.