Superior antitumor immune response achieved with proton over photon immunoradiotherapy is amplified by the nanoradioenhancer NBTXR3.

Recent findings suggest that immunoradiotherapy (IRT), combining photon radiotherapy (XRT) or proton radiotherapy (PRT) with immune checkpoint blockade, can enhance systemic tumor control. However, the comparative efficacy of XRT and PRT in IRT remains understudied. To address this, we compared outcomes between XRT + αPD1 and PRT + αPD1 in murine αPD1-resistant lung cancer (344SQR). We also assessed the impact of the nanoparticle radioenhancer NBTXR3 on both XRT + αPD1 and PRT + αPD1 for tumor control and examined the tumor immune microenvironment using single-cell RNA sequencing (scRNAseq). Additionally, mice cured by NBTXR3 + PRT + αPD1 were rechallenged with three lung cancer cell lines to evaluate memory antitumor immunity. PRT + αPD1 showed superior local tumor control and abscopal effects compared to XRT + αPD1. NBTXR3 + PRT + αPD1 significantly outperformed NBTXR3 + XRT + αPD1 in tumor control, promoting greater infiltration of antitumor lymphocytes into irradiated tumors. Unirradiated tumors treated with NBTXR3 + PRT + αPD1 had more NKT cells, CD4 T cells, and B cells, with fewer Tregs, than those treated with NBTXR3 + XRT + αPD1. NBTXR3 + PRT + αPD1 also stimulated higher expression of IFN-γ, GzmB, and Nkg7 in lymphocytes, reduced the TGF-ß pathway, and increased tumor necrosis factor alpha expression compared to NBTXR3 + XRT + αPD1. Moreover, NBTXR3 + PRT + αPD1 resulted in greater M1 macrophage polarization in both irradiated and unirradiated tumors. Mice achieving remission through NBTXR3 + PRT + αPD1 exhibited a robust memory immune response, effectively inhibiting growth of subsequent tumors from three distinct lung cancer cell lines. Proton IRT combined with NBTXR3 offers enhanced tumor control and survival rates over photon-based treatments in managing αPD1-resistant lung cancer, indicating its potential as a potent systemic therapy.

Combining a nanoparticle-mediated immunoradiotherapy with dual blockade of LAG3 and TIGIT improves the treatment efficacy in anti-PD1 resistant lung cancer.

BACKGROUND: While improvements in immunoradiotherapy have significantly improved outcomes for cancer patients, this treatment approach has nevertheless proven ineffective at controlling the majority of malignancies. One of the mechanisms of resistance to immunoradiotherapy is that immune cells may be suppressed via the myriad of different immune checkpoint receptors. Therefore, simultaneous blockade of multiple immune checkpoint receptors may enhance the treatment efficacy of immunoradiotherapy. METHODS: We combined NBTXR3-enhanced localized radiation with the simultaneous blockade of three different checkpoint receptors: PD1, LAG3, and TIGIT, and tested the treatment efficacy in an anti-PD1-resistant lung cancer model in mice. 129 Sv/Ev mice were inoculated with fifty thousand αPD1-resistant 344SQR cells in the right leg on day 0 to establish primary tumors and with the same number of cells in the left leg on day 4 to establish the secondary tumors. NBTXR3 was intratumorally injected into the primary tumors on day 7, which were irradiated with 12 Gy on days 8, 9, and 10. Anti-PD1 (200 µg), αLAG3 (200 µg), and αTIGIT (200 µg) were given to mice by intraperitoneal injections on days 5, 8, 11, 14, 21, 28, 35, and 42. RESULTS: This nanoparticle-mediated combination therapy is effective at controlling the growth of irradiated and distant unirradiated tumors, enhancing animal survival, and is the only one that led to the destruction of both tumors in approximately 30% of the treated mice. Corresponding with this improved response is robust activation of the immune response, as manifested by increased numbers of immune cells along with a transcriptional signature of both innate and adaptive immunity within the tumor. Furthermore, mice treated with this combinatorial therapy display immunological memory response when rechallenged by the same cancer cells, preventing tumor engraftment. CONCLUSION: Our results strongly attest to the efficacy and validity of combining nanoparticle-enhanced radiotherapy and simultaneous blockade of multiple immune checkpoint receptors and provide a pre-clinical rationale for investigating its translation into human patients.

A multicenter open-label randomized phase II trial of paclitaxel plus EP-100, a novel LHRH receptor-targeted, membrane-disrupting peptide, versus paclitaxel alone for refractory or recurrent ovarian cancer.

OBJECTIVE: This randomized open-label phase II study evaluated the safety and clinical activity of EP-100 plus weekly paclitaxel in patients with recurrent ovarian cancer expressing positive LHRH receptor. METHODS: In a limited "run-in" dose escalation phase for EP-100, six patients were treated with ascending dose levels (13 mg/m2, 20 mg/m2, 30 mg/m2). In the randomized phase, patients received weekly paclitaxel (80 mg/m2 intravenously) plus twice weekly EP-100 (30 mg/m2 intravenously; combination arm) or weekly paclitaxel alone (80 mg/m2 intravenously; paclitaxel arm). The primary study endpoint was overall response rate (ORR). RESULTS: Forty-four patients were then randomized to either the experimental combination arm (n = 23) or the standard of care paclitaxel monotherapy arm (n = 21). The ORR was 35% (95%CI 16%-57%) for the combination arm and 33% (95% CI 15%-57%) for the paclitaxel arm. An interesting observation from an unplanned analysis was that a subset of patients with target liver lesions showed a greater overall response rate to the combination (69%) compared to paclitaxel alone (16%). The frequency of treatment-related grade 3-4 adverse events was similar between treatment arms: 48% vs 43% for the combination and paclitaxel arms, respectively. CONCLUSIONS: ORR in the EP-100 combination arm was similar to that in the group treated with paclitaxel alone; however, a subset of patients with liver metastases appeared to benefit from the combination. The addition of EP-100 did not appear to augment the adverse event profile of paclitaxel and was well tolerated.

Inhibition of MER proto-oncogene tyrosine kinase by an antisense oligonucleotide enhances treatment efficacy of immunoradiotherapy.

BACKGROUND: The combination of radiotherapy and immunotherapy (immunoradiotherapy) has been increasingly used for treating a wide range of cancers. However, some tumors are resistant to immunoradiotherapy. We have previously shown that MER proto-oncogene tyrosine kinase (MerTK) expressed on macrophages mediates resistance to immunoradiotherapy. We therefore sought to develop therapeutics that can mitigate the negative impact of MerTK. We designed and developed a MerTK specific antisense oligonucleotide (ASO) and characterized its effects on eliciting an anti-tumor immune response in mice. METHODS: 344SQR cells were injected into the right legs on day 0 and the left legs on day 4 of 8-12 weeks old female 129sv/ev mice to establish primary and secondary tumors, respectively. Radiation at a dose of 12 Gy was given to the primary tumors on days 8, 9, and 10. Mice received either anti-PD-1, anti-CTLA-4 or/and MerTK ASO starting from day 1 post tumor implantation. The composition of the tumor microenvironment and the level of MerTK on macrophages in the tumor were evaluted by flow cytometry. The expression of immune-related genes was investigated with NanoString. Lastly, the impact of MerTK ASO on the structure of the eye was histologically evaluated. RESULTS: Remarkably, the addition of MerTK ASO to XRT+anti-PD1 and XRT+anti-CTLA4 profoundly slowed the growth of both primary and secondary tumors and significantly extended survival. The ASO significantly reduced the expression of MerTK in tumor-associated macrophages (TAMs), reprograming their phenotype from M2 to M1. In addition, MerTK ASO increased the percentage of Granzyme B+ CD8+ T cells in the secondary tumors when combined with XRT+anti-CTLA4. NanoString results demonstrated that the MerTK ASO favorably modulated immune-related genes for promoting antitumor immune response in secondary tumors. Importantly, histological analysis of eye tissues demonstrated that unlike small molecules, the MerTK ASO did not produce any detectable pathology in the eyes. CONCLUSIONS: The MerTK ASO can significantly downregulate the expression of MerTK on TAMs, thereby promoting antitumor immune response. The combination of MerTK ASO with immunoradiotherapy can safely and significantly slow tumor growth and improve survival.

Enhanced tumor control and survival in preclinical models with adoptive cell therapy preceded by low-dose radiotherapy.

Introduction: Effective infiltration of chimeric antigen receptor T (CAR-T) cells into solid tumors is critical for achieving a robust antitumor response and improving therapeutic outcomes. While CAR-T cell therapies have succeeded in hematologic malignancies, their efficacy in solid tumors remains limited due to poor tumor penetration and an immunosuppressive tumor microenvironment. This study aimed to evaluate the potential of low-dose radiotherapy (LDRT) administered before T-cell therapy to enhance the antitumor effect by promoting CAR-T cell infiltration. We hypothesized that combining LDRT with T-cell therapy would improve tumor control and survival compared to either treatment alone. Methods: We investigated this hypothesis using two NSG mouse models bearing GSU or CAPAN-2 solid tumors. The mice were treated with engineered CAR-T cells targeting guanyl cyclase-C (GCC) or mesothelin as monotherapy or in combination with LDRT. Additionally, we extended this approach to a C57BL/6 mouse model implanted with MC38-gp100+ cells, followed by adoptive transfer of pmel+ T cells before and after LDRT. Tumor growth and survival outcomes were monitored in all models. Furthermore, we employed atomic force microscopy (AFM) in a small cohort to assess the effects of radiotherapy on tumor stiffness and plasticity, exploring the role of tumor nanomechanics as a potential biomarker for treatment efficacy. Results: Our results demonstrated enhanced tumor control and prolonged survival in mice treated with LDRT followed by T-cell therapy across all models. The combination of LDRT with CAR-T or pmel+ T-cell therapy led to superior tumor suppression and survival compared to monotherapy, highlighting the synergistic impact of the combined approach. Additionally, AFM analysis revealed significant changes in tumor stiffness and plasticity in response to LDRT, suggesting that the nanomechanical properties of the tumor may be predictive of therapeutic response. Discussion: The findings of this study highlight the transformative potential of incorporating LDRT as a precursor to adoptive T-cell therapy in solid tumors. By promoting CAR-T and pmel+ T-cell infiltration into the tumor microenvironment, LDRT enhanced tumor control and improved survival outcomes, offering a promising strategy to overcome the challenges associated with CAR-T therapy in solid tumors. Additionally, the changes in tumor nanomechanics observed through AFM suggest that tumor stiffness and plasticity could be biomarkers for predicting treatment outcomes. These results support further investigation into the clinical application of this combined approach to improve the efficacy of cell-based therapies in patients with solid tumors.

Molecular MRI for sensitive and specific detection of lung metastases.

Early and specific detection of metastatic cancer cells in the lung (the most common organ targeted by metastases) could significantly improve cancer treatment outcomes. However, the most widespread lung imaging methods use ionizing radiation and have low sensitivity and/or low specificity for cancer cells. Here we address this problem with an imaging method to detect submillimeter-sized metastases with molecular specificity. Cancer cells are targeted by iron oxide nanoparticles functionalized with cancer-binding ligands, then imaged by high-resolution hyperpolarized (3)He MRI. We demonstrate in vivo detection of pulmonary micrometastates in mice injected with breast adenocarcinoma cells. The method not only holds promise for cancer imaging but more generally suggests a fundamentally unique approach to molecular imaging in the lungs.

Durable control of metastases in an HLA-A2+ patient with refractory melanoma after low-dose radiotherapy in combination with MAGE-A4 T cell therapy: a case report.

There is no currently approved adoptive cellular therapy for solid tumors. Pre-clinical and clinical studies have demonstrated that low-dose radiotherapy (LDRT) can enhance intratumoral T cell infiltration and efficacy. This case report describes a 71-year-old female patient with rectal mucosal melanoma that had developed metastases to liver, lung, mediastinum, axillary nodes, and brain. After systemic therapies had failed, she enrolled in the radiation sub-study of our phase-I clinical trial exploring the safety and efficacy of afamitresgene autoleucel (afami-cel), genetically engineered T cells with a T cell receptor (TCR) targeting the MAGE-A4 tumor antigen in patients with advanced malignancies (NCT03132922). Prior to the infusion of afami-cel, she received concurrent lymphodepleting chemotherapy and LDRT at 5.6 Gy/4 fractions to the liver. Time to partial response was 10 weeks, and duration of overall response was 18.4 weeks. Although the patient progressed at 28 weeks, the disease was well controlled after high-dose radiotherapy to liver metastases and checkpoint inhibitors. As of the last follow-up, she remains alive over two years after LDRT and afami-cel therapy. This report suggests that afami-cel in combination with LDRT safely enhanced clinical benefit. This provides evidence for further exploring the benefit of LDRT in TCR-T cell therapy.

Inhibition of STAT6 with Antisense Oligonucleotides Enhances the Systemic Antitumor Effects of Radiotherapy and Anti-PD-1 in Metastatic Non-Small Cell Lung Cancer.

Diverse factors contribute to the limited clinical response to radiotherapy (RT) and immunotherapy in metastatic non-small cell lung cancer (NSCLC), among which is the ability of these tumors to recruit a retinue of suppressive immune cells-such as M2 tumor-associated macrophages (TAM)-thereby establishing an immunosuppressive tumor microenvironment that contributes to tumor progression and radio resistance. M2 TAMs are activated by the STAT6 signaling pathway. Therefore, we targeted STAT6 using an antisense oligonucleotide (ASO) along with hypofractionated RT (hRT; 3 fractions of 12 Gy each) to primary tumors in three bilateral murine NSCLC models (Lewis lung carcinoma, 344SQ-parental, and anti-PD-1-resistant 344SQ lung adenocarcinomas). We found that STAT6 ASO plus hRT slowed growth of both primary and abscopal tumors, decreased lung metastases, and extended survival. Interrogating the mechanism of action showed reduced M2 macrophage tumor infiltration, enhanced TH1 polarization, improved T-cell and macrophage function, and decreased TGFß levels. The addition of anti-PD-1 further enhanced systemic antitumor responses. These results provide a preclinical rationale for the pursuit of an alternative therapeutic approach for patients with immune-resistant NSCLC.

Nanoparticle-enhanced proton beam immunoradiotherapy drives immune activation and durable tumor rejection.

The combination of radiation therapy (RT) and immunotherapy has emerged as a promising treatment option in oncology. Historically, x-ray radiation (XRT) has been the most commonly used form of RT. However, proton beam therapy (PBT) is gaining recognition as a viable alternative, as it has been shown to produce similar outcomes to XRT while minimizing off-target effects. The effects of PBT on the antitumor immune response have only just begun to be described, and to our knowledge no studies to date have examined the effect of PBT as part of a combinatorial immunoradiotherapeutic strategy. Here, using a 2-tumor model of lung cancer in mice, we show that PBT in tandem with an anti-PD1 antibody substantially reduced growth in both irradiated and unirradiated tumors. This was accompanied by robust activation of the immune response, as evidenced by whole-tumor and single-cell RNA sequencing showing upregulation of a multitude of immune-related transcripts. This response was further significantly enhanced by the injection of the tumor to be irradiated with NBTXR3 nanoparticles. Tumors of mice treated with the triple combination exhibited increased infiltration and activation of cytotoxic immune cells. This triple combination eradicated both tumors in 37.5% of the treated mice and showed robust long-term immunity to cancer.

NBTXR3 improves the efficacy of immunoradiotherapy combining nonfucosylated anti-CTLA4 in an anti-PD1 resistant lung cancer model.

The efficacy of immunoradiotherapy consisting of radiation therapy and immune checkpoint blockade relies on effectively promoting the systemic antitumor immune response's activation while simultaneously reducing local factors favoring immune suppression. We previously demonstrated that NBTXR3, a nanoparticle radioenhancer, significantly improved immune responses in a murine anti-PD1-resistant metastatic lung cancer model. We hypothesize that radioactivated-NBTXR3 addition to anti-PD1 and a second-generation anti-CTLA4 could improve treatment effectiveness. To test this hypothesis, we inoculated mice with 344SQR cells in the right and left legs to establish primary and secondary tumors. The primary tumors were intratumorally injected with NBTXR3 nanoparticles on day 7, followed by three fractions of 12 Gy radiation on days 8, 9, and 10. The secondary tumors received two fractions of 1Gy radiation on days 13 and 14. Multiple rounds of anti-PD1, anti-CTLA4 or nonfucosylated anti-CTLA4 were given to the mice. Immune profiling of the tumors revealed that the combination of NBTXR3 with immunoradiotherapy significantly upregulated the activities of a wide range of antitumor immune pathways and reduced the abundance of regulatory suppressor T cells. This combination effectively eradicated the primary and secondary tumors and increased animal survival to 75%. Remarkably, previously treated with NBTXR3-containing treatment, the survivor mice exhibited a long-lasting antitumor memory immune response. This data provides compelling evidence of the efficacy of NBTXR3 to synergize with the immunoradiotherapy approach when combined with an anti-PD1 and multiple checkpoints such as a second generation anti-CTLA4 and show the potential for clinical uses of antitumor immunomodulatory effects of NBTXR3.

Novel Use of Low-Dose Radiotherapy to Modulate the Tumor Microenvironment of Liver Metastases.

Despite multiple therapeutic approaches, the presence of liver metastases carries a guarded prognosis, urgently necessitating further clinical and scientific research to develop curative interventions. The liver is an immunoprivileged organ that suppresses the effectiveness of immunotherapies in patients with hepatic metastases. Cancer immunotherapies have been successfully bolstered by low-dose radiotherapy (LDRT), which is capable of reprogramming the tumor microenvironment (TME) from an immunosuppressive to an immunostimulatory one. Likewise, LDRT may be able to revoke the immune privilege enjoyed by the liver, permitting successful immunotherapies there. Here, we first review challenges that face the treatment of liver metastases. We next outline emerging preclinical and clinical evidence supporting enhanced systemic tumor control of LDRT in the context of cancer immunotherapy. Finally, we will discuss the rationale of combining liver-directed LDRT with immunostimulatory strategies to overcome immune resistance and achieve better clinical response. This notion is supported by a recent case study in which a patient who had progressed following T cell therapy experienced a complete response after LDRT to the liver.

Enhanced Immunotherapy with LHRH-R Targeted Lytic Peptide in Ovarian Cancer.

Here, we examined the role of EP-100 [luteinizing hormone-releasing hormone (LHRH) ligand joined to a lytic peptide], improving the efficacy of immune checkpoint blockade. LHRH-R-positive murine ovarian cancer cells (ID8, IG10, IF5, and 2C12) were sensitive to EP-100 and were specifically killed at low micromolar levels through LHRH-R. EP-100 increased PD-L1 levels on murine ovarian cancer cells. In vivo syngeneic mouse models (ID8 and IG10) demonstrated that single-agent EP-100 reduced tumor volume, tumor weight, and ascites volume. The greatest reductions in tumor and ascites volume were observed with the combination of EP-100 with an anti-PD-L1 antibody. Immune profiling analysis showed that the population of CD8+ T cells, natural killer cells, dendritic cells, and macrophages were significantly increased in tumor and ascitic fluid samples treated with anti-PD-L1, EP-100, and the combination. However, monocytic myeloid suppressor cells, B cells, and regulatory T cells were decreased in tumors treated with anti-PD-L1, EP-100, or the combination. In vitro cytokine arrays revealed that EP-100 induced IL1α, IL33, CCL20, VEGF, and Low-density lipoprotein receptor (LDLR) secretion. Of these, we validated increasing IL33 levels following EP-100 treatment in vitro and in vivo; we determined the specific biological role of CD8+ T-cell activation with IL33 gene silencing using siRNA and Cas9-CRISPR approaches. In addition, we found that CD8+ T cells expressed very low level of LHRH-R and were not affected by EP-100. Taken together, EP-100 treatment had a substantial antitumor efficacy, particularly in combination with an anti-PD-L1 antibody. These results warrant further clinical development of this combination.

iDQC anisotropy map imaging for tumor tissue characterization in vivo.

Intermolecular double quantum coherences (iDQCs), signals that result from simultaneous transitions of two or more separated spins, are known to produce images that are highly sensitive to subvoxel structure, particularly local anisotropy. Here we demonstrate how iDQCs signal can be used to efficiently detect the anisotropy created in breast tumor tissues and prostate tumor tissues by targeted (LHRH-conjugated) superparamagnetic nanoparticles (SPIONs), thereby distinguishing the necrotic area from the surrounding tumor tissue.

GnRH-R-Targeted Lytic Peptide Sensitizes BRCA Wild-type Ovarian Cancer to PARP Inhibition.

EP-100 is a synthetic lytic peptide that specifically targets the gonadotropin-releasing hormone receptor on cancer cells. To extend the utility of EP-100, we aimed to identify effective combination therapies with EP-100 for ovarian cancer and explore potential mechanisms of this combination. A series of in vitro (MTT assay, immunoblot analysis, reverse-phase protein array, comet assay, and immunofluorescence staining) and in vivo experiments were carried out to determine the biological effects of EP-100 alone and in combination with standard-of-care drugs. EP-100 decreased the viability of ovarian cancer cells and reduced tumor growth in orthotopic mouse models. Of five standard drugs tested (cisplatin, paclitaxel, doxorubicin, topotecan, and olaparib), we found that the combination of EP-100 and olaparib was synergistic in ovarian cancer cell lines. Further experiments revealed that combined treatment of EP-100 and olaparib significantly increased the number of nuclear foci of phosphorylated histone H2AX. In addition, the extent of DNA damage was significantly increased after treatment with EP-100 and olaparib in comet assay. We performed reverse-phase protein array analyses and identified that the PI3K/AKT pathway was inhibited by EP-100, which we validated with in vitro experiments. In vivo experiment using the HeyA8 mouse model demonstrated that mice treated with EP-100 and olaparib had lower tumor weights (0.06 ± 0.13 g) than those treated with a vehicle (1.19 ± 1.09 g), EP-100 alone (0.62 ± 0.78 g), or olaparib alone (0.50 ± 0.63 g). Our findings indicate that combining EP-100 with olaparib is a promising therapeutic strategy for ovarian cancer.

Pharmacokinetics and pharmacodynamics of Phor21-betaCG(ala), a lytic peptide conjugate.

Phor21-betaCG(ala), a 36-amino acid peptide comprised of a lytic peptide (Phor21) conjugated to a modified 15-amino acid segment of the beta-chain of chorionic gonadotropin (betaCG(ala)), selectively kills cancer cells that over-express luteinizing hormone/chorionic gonadotropin (LH/CG) receptors by disrupting cellular membrane structure. These studies were designed to further characterize its in-vitro inhibition and in-vivo destruction of prostate cancer cells, biostability and pharmacokinetics to determine its pharmacokinetic and pharmacodynamic profile. Inhibitory effects of Phor21-betaCG(ala) were tested in PC-3 and Caco-2 cells as well as in nude mice bearing PC-3 cells transfected with the luciferase gene (PC-3.luc). Plasma stability, protease hydrolysis and pharmacokinetics of Phor21-betaCG(ala) were measured by using liquid chromatography mass spectrometry (LC/MS/MS). Phor21-betaCG(ala) selectively inhibited proliferation in-vitro and in-vivo metastases of PC-3 cells. Phor21-betaCG(ala) was relatively stable in mouse, rat, dog and human plasma. Its degradation was partially due to protease hydrolysis and thermodynamic catalysis. Intravenous administration of Phor21-betaCG(ala) showed its blood C(max) and AUC(0-->infinity) around the in-vitro effective levels. In the tested rodents, Phor21-betaCG(ala) displayed a moderate volume of distribution at steady state (Vd(ss)) and slow clearance (Cl) in the rodents. In conclusion, Phor21-betaCG(ala) displayed promising in-vitro and in-vivo anti-cancer activity with favourable pharmacokinetics, and may offer a novel approach to metastatic cancer chemotherapy.

Effective treatment of human breast tumor in a mouse xenograft model with herpes simplex virus type 1 specifying the NV1020 genomic deletion and the gBsyn3 syncytial mutation enabling high viral replication and spread in breast cancer cells.

A new oncolytic and fusogenic herpes simplex virus type 1 (HSV-1) was constructed on the basis of the wildtype HSV-1(F) strain. To provide for safety and tumor selectivity, the virus carried a large deletion including one of the two alpha4, gamma(1)34.5, alpha0 genes and the latency-associated transcript region. The gamma(1)34.5 gene, a major neurovirulence factor, was replaced by a gene cassette constitutively expressing the red fluorescent protein gene. Homologous recombination was used to transfer the fusogenic gBsyn3 mutation to the viral genome to produce the OncSyn virus. OncSyn causes extensive virus-induced cell fusion (syncytia) and replicates to higher titers than the parental Onc and HSV-1(F) strains in breast cancer cells. Biochemical analysis revealed that the OncSyn virus retains a stable genome and expresses all major viral glycoproteins. A xenograft mouse model system using MDA-MB-435S-luc (MM4L) human breast cancer cells constitutively expressing the luciferase gene implanted within the interscapular region of animals was used to test the ability of the virus to inactivate breast tumor cells in vivo. Seventy-two mice bearing MM4L breast cancer xenografts were randomly divided into three groups and given two rounds of three consecutive intratumoral injections of OncSyn, inactivated OncSyn, or phosphate-buffered saline 3 days apart. A single round of virus injections resulted in a drastic reduction of tumor sizes (p

Destruction of breast cancers and their metastases by lytic peptide conjugates in vitro and in vivo.

In a series of in vivo and in vitro experiments, the concept has been established that breast cancer cells that express LH/CG or LHRH receptors can be targeted and destroyed by constructs consisting of a lytic peptide moiety and a 15-amino acid segment of the beta-chain of CG or by an LHRH lytic peptide conjugate. Data obtained in vitro established the validity of this concept, showed the specificities of the Hecate-betaCG, and Phor14 and Phor21-betaCG conjugates in killing cells that express functional LH/CG receptors and proved that the LH/CG receptor capacity is directly related to the compound's specificity. In in vivo experiments, Hecate-betaCG, Phor14-betaCG, and Phor21-betaCG(ala) each caused highly significant reductions of tumor volume and tumor burden in nude mice bearing breast cancer xenografts; Hecate and Phor21 alone or conjugated with non-specific peptides were not effective. Most importantly, the lytic peptide conjugates were all highly effective in targeting and destroying disseminated breast cancer metastases in lymph nodes, bones, lungs and other organs.

Conjugates of lytic peptides and LHRH or betaCG target and cause necrosis of prostate cancers and metastases.

In a series of in vivo and in vitro experiments, it was shown that membrane disrupting lytic peptides (Hecate, Phor14, or Phor21) conjugated to a 15 amino acid segment of the beta chain of CG or to LHRH were able to target and destroy hormone dependent and independent human prostate cancer xenografts in nude mice. In vitro sensitivity of the cells to the drugs was directly related to LH/CG receptor expression, and pretreatment in vitro or in vivo with estrogens or FSH to enhance LH/CG receptor expression capacity and increased sensitivity to the drugs. Administration of unconjugated Hecate and LHRH was ineffective. Most importantly, all of the lytic peptide-betaCG conjugates tested were highly effective in destroying prostate cancer metastatic cells in lymph nodes, bones and lungs.

Sub-cellular accumulation of magnetic nanoparticles in breast tumors and metastases.

In this study, the sub-cellular accumulation of superparamagnetic iron oxide nanoparticles (SPIONs) in breast tumors and peripheral organs were investigated. MNPs were conjugated with luteinizing hormone releasing hormone (LHRH), whose receptors are expressed by most types of breast cancer cells. After the nanoparticles were injected into female nude mice bearing MDA-MB-435S.luc tumors, the mice were sacrificed to collect tumors and peripheral organs for biological and TEM analyses. LHRH conjugated SPIONs (LHRH- SPIONs) were found to accumulate in cancer cells, mainly in the primary tumors and the metastatic lungs, where they aggregated to form clusters. In contrast, most of the unconjugated SPIONs were collected in the liver cells. The results suggest that LHRH- SPIONs can be used to target cancer cells in the primary breast tumors and the lung metastases. TEM is also shown to be a useful tool for the studies of sub-cellular distributions of SPIONs in tumors and tissues.

Membrane disrupting lytic peptides for cancer treatments.

Membrane disrupting lytic peptides are abundant in nature and serve insects, invertebrates, vertebrates and humans as defense molecules. Initially, these peptides attracted attention as antimicrobial agents; later, the sensitivity of tumor cells to lytic peptides was discovered. In the last decade intensive research has been conducted to determine how lytic peptides lyse bacteria and tumor cells. A number of synthetic peptides have been designed to optimize their antibiotic and anti-tumor properties and improve their therapeutic capabilities. The sequences of alpha-helical cationic membrane disrupting peptides has been discussed, their proposed mechanisms of action reviewed, and their roles in cell selectivity and tumor cell destruction considered. Parameters important for the selection and design of lytic peptides for cancer treatments include increased activities against tumor cells, low cytolytic activities to normal mammalian cells and erythrocytes. The conjugation of lytic peptides with hormone ligands and the production of pro-peptides provide methods for targeting of cancer cells. The therapeutic possibilities in cancer treatment by targeted lytic peptides are broad and offer improvement to currently used chemotherapeutical drugs. Lytic peptides interact with the tumor cell membrane within minutes, and their activity is independent of multi-drug resistance. Lytic peptide-chorionic gonadotropin (CG) conjugates destroy primary tumors, prevent metastases and kill dormant and metastatic tumor cells. These conjugates do not destroy vital organs; they are not antigenic, and are more toxic to tumor cells than to non-malignant cells.