Efficacy and Safety of Chidamide in Combination with PD-1 Inhibitor and Radiotherapy for HER2-Negative Advanced Breast Cancer: Study Protocol of a Single Arm Prospective Study.

Purpose: As one of the most important breakthroughs in cancer therapy, immune checkpoint inhibitors have greatly prolonged survival of patients with breast cancer. However, their application and efficacy are limited, especially for advanced HER2-negative breast cancer. It has been reported that epigenetic modulation of the histone deacetylase (HDAC) inhibitor chidamide, as well as immune microenvironment modulation of radiotherapy are potentially synergistic with immunotherapy. Thus, the combination of chidamide, radiotherapy and immunotherapy is expected to improve prognosis of patients with advanced HER2-negative breast cancer. Patients and Methods: This is a single-arm, open, prospective clinical trial investigating the efficacy and safety of the combination of HDAC inhibitor chidamide, anti-PD-1 antibody sintilimab, and the novel immuno-radiotherapy, which aims to enhance efficacy of immunotherapy, in subsequent lines of therapy of HER2-negative breast cancer. Our study will include 35 patients with advanced breast cancer that has failed endocrine therapy and first-line chemotherapy. Participants will receive 30 mg of chidamide twice a week, 200 mg of sintilimab once every 3 weeks, combined with immuno-radiotherapy. Radiotherapy will be centrally 8 Gy for at least one lesion, and at least 1 Gy for the other lesions. We will complete three fractions of radiotherapy in one cycle. The primary endpoint is progression-free survival, and secondary endpoints are objective response rate, disease control rate and safety. Moreover, biomarkers including cytokines and lymphocyte subgroups will be explored. Conclusion: As a single-arm clinical trial, the analysis of the influence of each single treatment is limited. Besides, our study is an open study, which involves neither randomization nor blinding. In spite of the abovementioned limitations, this prospective clinical trial will give an insight into subsequent lines of therapy of HER2-negative advanced breast cancer, prolong the survival or achieve long remission for these participants, and identify potential responders.

Expression of four cancer-testis antigens in TNBC indicating potential universal immunotherapeutic targets.

OBJECTIVE: Immunotherapy is an attractive treatment for breast cancer. Cancer-testis antigens (CTAs) are potential targets for immunotherapy for their restricted expression. Here, we investigate the expression of CTAs in breast cancer and their value for prognosis. So as to hunt for a potential panel of CTAs for universal immunotherapeutic targets. MATERIAL AND METHODS: A total of 137 breast cancer tissue specimens including 51 triple-negative breast cancer (TNBC) were assessed for MAGE-A4, MAGEA1, NY-ESO-1, KK-LC-1 and PRAME expression by immunohistochemistry. The expression of PD-L1 and TILs was also calculated and correlated with the five CTAs. Clinical data were collected to evaluate the CTA's value for prognosis. Data from the K-M plotter were used as a validation cohort. RESULTS: The expression of MAGE-A4, NY-ESO-1 and KK-LC-1 in TNBC was significantly higher than in non-TNBC (P = 0.012, P = 0.005, P < 0.001 respectively). 76.47% of TNBC expressed at least one of the five CTAs. Patients with positive expression of either MAGE-A4 or PRAME had a significantly extended disease-free survival (DFS). Data from the Kaplan-Meier plotter confirm our findings. CONCLUSIONS: MAGE-A4, NY-ESO-1, PRAME and KK-LC-1 are overexpressed in breast cancer, especially in TNBC. Positive expression of MAGE-A4 or PARME may be associated with prolonged DFS. A panel of CTAs is attractive universal targets for immunotherapy.

Case Report: Small intestinal metastatic breast cancer: A case report and literature review.

Breast cancer is considered a malignant tumor with the highest incidence among women and is prone to develop distant metastasis. Small intestinal metastasis of breast cancer, however, is relatively rare. This case report describes a 49-year-old Chinese female patient who presented with small intestinal obstruction and was diagnosed with lobular breast cancer with small intestinal and contralateral breast metastasis. Clinical manifestations, clinicopathological features and potential mechanisms of metastasis, along with diagnosis and treatment, are discussed with a review of the relevant literature. Although small intestinal metastasis is rare in breast cancer, we should keep high alert on the possibility of gastrointestinal metastasis when treating lobular breast cancer patients.

Recent Progress on Therapeutic Vaccines for Breast Cancer.

Breast cancer remains the most frequently diagnosed malignancy worldwide. Advanced breast cancer is still an incurable disease mainly because of its heterogeneity and limited immunogenicity. The great success of cancer immunotherapy is paving the way for a new era in cancer treatment, and therapeutic cancer vaccination is an area of interest. Vaccine targets include tumor-associated antigens and tumor-specific antigens. Immune responses differ in different vaccine delivery platforms. Next-generation sequencing technologies and computational analysis have recently made personalized vaccination possible. However, only a few cases benefiting from neoantigen-based treatment have been reported in breast cancer, and more attention has been given to overexpressed antigen-based treatment, especially human epidermal growth factor 2-derived peptide vaccines. Here, we discuss recent advancements in therapeutic vaccines for breast cancer and highlight near-term opportunities for moving forward.

Lymph node-targeted neoantigen nanovaccines potentiate anti-tumor immune responses of post-surgical melanoma.

BACKGROUND: Neoantigens are considered ideal targets for immunotherapy, especially tumor vaccine, because of their strong specificity and immunogenicity. Here, we developed a neoantigen nanovaccine used liposomes with lymph-node targeting characteristic. METHODS: Our nanovaccine was composed of neoantigens, an amphiphilic liposome and an adjuvant Montanide™ ISA 51. Small animal imaging system and immunofluorescence staining were used to identify the distribution of nanovaccines. A subcutaneous-tumor-resection mouse model of melanoma was established to evaluate the anti-tumor efficacy. Flow cytometry was performed to assay the immune responses initiated by nanovaccines. RESULTS: Nanovaccines could traffic to lymph nodes, be uptaken by CD11c+ DCs and promote DCs maturity. After the treatment of our neoantigen nanovaccines, the average recurrence time was extended from 11 to 16 days and the median survival time was even prolonged 7.5 days relative to the control group (NS group). Nanovaccines increased neoantigen-specific T cells to 10-fold of free vaccines, and upregulated Th1 cytokines, such as IFN-γ and TNF-α. The anti-tumor activity of spleen lymphocytes in the nanovaccine group was significantly stronger than that of other groups. However, some immune-inhibitory cells or molecules in tumor microenvironment have been detected upregulated under the immune pressure of neoantigen nanovaccines, such as Tregs and PD-L1. The efficacy of the neoantigen nanovaccine combined with anti-PD1 antibody or Treg inhibiting peptide P60 was better than that of the single treatment. CONCLUSIONS: We developed a general vaccine strategy, triggering specific T cell responses, and provided feasible combination strategies for better anti-tumor efficacy.

Lipophilic recombinant-protein insertion endows lymphocytes with enhanced targeting-infiltration ability in EGFR positive cancer.

Adoptive T cell transfer is one of the most promising ways to combat solid tumors. However, the weak infiltration of T cells into tumor sites has restricted their antitumor efficacy. To overcome this obstacle, we used the lipophilic protein painting strategy to improve tumor targeting and penetrating capacity of lymphocytes for the first time. We synthesized the lipid anchor consisting of a bispecific recombinant protein iRGD-antiEGFR and DSPE-PEG derivates, then successfully inserted it into the membranes of T cells. This surface modification was non-invasive and could efficiently improve the infiltration ability of T cells into multicellular spheroids and tumor masses. The surface modified T cells also displayed superior antitumor activities in EGFR-positive tumor xenografts via systematic infusion. Moreover, the permeability and antitumor efficacy of these surface painted T cells could be remarkably enhanced when used in combination with local low-dose irradiation.

Hepatic Arterial Infusion Oxaliplatin Plus Oral S-1 Chemotherapy in Gastric Cancer with Unresectable Liver Metastases: A Case Series and Literature Review.

OBJECTIVE: The use of hepatic artery infusion (HAI) as a regional therapy against liver metastasis has rarely been reported in gastric cancer. This study aimed to evaluate the efficacy and safety of HAI oxaliplatin plus oral S-1 chemotherapy in first-line palliative therapy for gastric cancer with multiple liver metastases (GCLM). METHODS: We reviewed the records of five patients with GCLM who received HAI oxaliplatin (70-80 mg/m2 2 hrs d1,15) administered via a port-catheter system and S-1 with oral (35-40 mg/m2 twice daily for d1-14, 28 days for one cycle). Follow-up examination and efficacy evaluation were executed periodically. RESULTS: Until the 4th cycle response evaluation, the local effective rate and control rate were 40% and 80%, respectively; only one patient developed progression. HAI chemotherapy had a better local control against liver metastases (median progression-free survival: hepatic, 8.8 months vs. extrahepatic, 6.2 months), accompanied by less systemic toxicity, decreased tumour markers and symptomatic relief. CONCLUSION: HAI oxaliplatin plus oral S-1 chemotherapy can be considered as a new choice of first-line treatment for GCLM, which is also a good approach for controlling extrahepatic lesions with less adverse events.

Novel Hsp90 Inhibitor C086 Potently Inhibits Non-Small Cell Lung Cancer Cells As A Single Agent Or In Combination With Gefitinib.

PURPOSE: Inhibition of heat shock protein 90 (Hsp90) can lead to degradation of multiple client proteins, which are involved in tumor progression. Elevated Hsp90 expression has been linked to poor prognosis in patients with non-small cell lung cancer (NSCLC). Discovery of effective drug is a promising strategy to improve patient survival. This study aims to investigate the synergistic antitumor mechanism of C086 combined with gefitinib in NSCLC cells in vitro. METHODS: The binding of C086, gefitinib, and the combinations to Hsp90 was characterized by fluorescence quenching experiments. The inhibition of A549 or NCI-H1975 cell proliferation and apoptosis by C086 and gefitinib as a single agent or in combinations were performed using CFSE staining assays, AnnexinV-APC/PI and Western blot. RESULTS: C086 alone or with gefitinib reduces proliferation and increases proapoptotic caspase activation of both wild-type and mutation NSCLC, with NCI-H1975 cells showing much greater sensitivity to C086 and the combinations than A549 cells. The combination of C086 and gefitinib showed synergistic reduction of EGFR expression and the downstream PI3K/Akt and Ras-Raf-Erk pathways enhanced suppression of Erk signaling. CONCLUSION: C086 combined gefitinib has a good synergistic antitumor effect in vitro. Therefore, the combination of C086 and gefitinib may provide a new theoretical basis and ideas for the treatment of NSCLC patients.

iRGD synergizes with PD-1 knockout immunotherapy by enhancing lymphocyte infiltration in gastric cancer.

Poor infiltration of activated lymphocytes into tumors represents a fundamental factor limiting the therapeutic effect of adoptive cell immunotherapy. A tumor-penetrating peptide, iRGD, has been widely used to deliver drugs into tumor tissues. In this study, we demonstrate for the first time that iRGD could also facilitate the infiltration of lymphocytes in both 3D tumor spheroids and several xenograft mouse models. In addition, combining iRGD modification with PD-1 knockout lymphocytes reveals a superior anti-tumor efficiency. Mechanistic studies demonstrate that the binding of iRGD to neuropilin-1 results in tyrosine phosphorylation of the endothelial barrier regulator VE-cadherin, which plays a role in the opening of endothelial cell contacts and the promotion of transendothelial lymphocyte migration. In summary, these results demonstrate that iRGD modification could promote tumor-specific lymphocyte infiltration, and thereby overcome the bottleneck associated with adoptive immune cell therapy in solid tumors.

Lipid insertion enables targeted functionalization of paclitaxel-loaded erythrocyte membrane nanosystem by tumor-penetrating bispecific recombinant protein.

BACKGROUND: There is currently much interest in cancer cell targeting and tumor penetrating for research and therapeutic purposes. PURPOSE: To improve targeting delivery of antitumor drugs to gastric cancer, in this study, a tumor-targeting biocompatible drug delivery system derived from erythrocyte membrane for delivering paclitaxel (PTX) was constructed. METHODS: Erythrocyte membrane of human red blood cells (RBCs) were used for preparing of erythrocyte membrane-derived vesicles. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(maleimide[polyethylene glycol]-3400) (DSPE-PEG-MAL), a phospholipid derivative, was used to insert tumor-targeting molecular into erythrocyte membrane-derived vesicles. A lipid insertion method was used to functionalize these vesicles without the need for direct chemical conjugation. Furthermore, a tumor-penetrating bispecific recombinant protein named anti-EGFR-iRGD was used for the first time in this work to enable nanosystem to target and penetrate efficiently into the tumor site. RESULTS: Paclitaxel (PTX)-loaded anti-EGFR-iRGD-modified erythrocyte membrane nano-system (anti-EGFR-iRGD-RBCm-PTX, abbreviated to PRP) were manufactured. PRP was spheroid, uniformly size, about 171.7±4.7 nm in average, could be stable in vitro for 8 days, and released PTX in a biphasic pattern. PRP showed comparable cytotoxicity toward human gastric cancer cells in vitro. In vivo studies showed that, PRP accumulated in tumor site within 2 h of administration, lasted longer than 48 h, and the tumor volume was reduced 61% by PRP treatment in Balb/c nude mice, without causing severe side effects. CONCLUSION: PRP has potential applications in cancer treatment and as an adjunct for other anticancer strategies.

C1206, a novel curcumin derivative, potently inhibits Hsp90 and human chronic myeloid leukemia cells in vitro.

4-(4-Pyridinyl methylene) curcumin (C1206) is a new derivative of curcumin that is more active than curcumin in inhibition of heat shock protein 90 (Hsp90) and antitumor action. In this study we investigated the relationship between C1206-induced inhibition of Hsp90 and its anti-leukemic effects. The fluorescence quenching experiments showed that C1206 seemed to bind the middle dimerization domain of Hsp90. The interaction between C1206 and Hsp90 was driven mainly by electrostatic interaction. In in vitro enzyme activity assay, C1206 dose-dependently inhibited Hsp90 ATPase activity with an IC50 value of 4.17 µmol/L. In both imatinib-sensitive K562 chronic myeloid leukemia cells and imatinib-resistant K562/G01 chronic myeloid leukemia cells, C1206 (0.4-3.2 µmol/L) dose-dependently caused the degradation of Hsp90 client proteins and downstream proteins (AKT, MEK, ERK, C-RAF, P-AKT, P-MEK and P-ERK). Furthermore, C1206 (0.4-3.2 µmol/L) dose-dependently induced apoptosis of K562 and K562/G01 cells through triggering mitochondrial pathway. Consistent with this result, C1206 inhibited the proliferation of K562 and K562/G01 cells with IC50 values of 1.10 and 0.60 µmol/L, respectively. These results suggest that C1206 is a novel Hsp90 inhibitor and a promising therapeutic agent for chronic myeloid leukemia.

Human cytotoxic T-lymphocyte membrane-camouflaged nanoparticles combined with low-dose irradiation: a new approach to enhance drug targeting in gastric cancer.

Cell membrane-derived nanoparticles are becoming more attractive because of their ability to mimic many features of their source cells. This study reports on a biomimetic delivery platform based on human cytotoxic T-lymphocyte membranes. In this system, the surface of poly-lactic-co-glycolic acid nanoparticles was camouflaged using T-lymphocyte membranes, and local low-dose irradiation (LDI) was used as a chemoattractant for nanoparticle targeting. The T-lymphocyte membrane coating was verified using dynamic light scattering, transmission electron microscopy, and confocal laser scanning microscopy. This new platform reduced nanoparticle phagocytosis by macrophages to 23.99% (P=0.002). Systemic administration of paclitaxel-loaded T-lymphocyte membrane-coated nanoparticles inhibited the growth of human gastric cancer by 56.68% in Balb/c nude mice. Application of LDI at the tumor site significantly increased the tumor growth inhibition rate to 88.50%, and two mice achieved complete remission. Furthermore, LDI could upregulate the expression of adhesion molecules in tumor vessels, which is important in the process of leukocyte adhesion and might contribute to the localization of T-lymphocyte membrane-encapsulated nanoparticles in tumors. Therefore, this new drug-delivery platform retained both the long circulation time and tumor site accumulation ability of human cytotoxic T lymphocytes, while local LDI could significantly enhance tumor localization.

sTRAIL-iRGD is a promising therapeutic agent for gastric cancer treatment.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills tumor cells and augments chemotherapeutics in vivo. Here, we developed sTRAIL-iRGD, a recombinant protein consisting of sTRAIL fused to CRGDKGPDC, a C-terminal end binding peptide with an integrin-binding arginine-glycine-aspartic acid (iRGD) motif. CRGDKGPDC is a tumor-homing peptide with high penetration into tumor tissue and cells. We found that sTRAIL-iRGD internalized into cultured gastric cancer tumor cells and localized to both the tumor mass in vivo and three-dimensional multicellular spheroids in vitro. sTRAIL-iRGD had an antitumor effect in tumor cell lines, multicellular spheroids and nude mice with tumors. Repeated treatment with sTRAIL-iRGD reduced tumor growth and volume in vivo. Mice treated with sTRAIL-iRGD and paclitaxel (PTX) in combination showed no sign of sTRAIL-iRGD-related liver toxicity. Our data suggest that sTRAIL-iRGD is a promising anti-gastric cancer agent with high selectivity and limited systemic toxicity.

Tumor-penetrating peptide fused to a pro-apoptotic peptide facilitates effective gastric cancer therapy.

KLA (sequence, KLAKLAKKLAKLAK) is a peptide which leads to programmed cell death by disrupting the mitochondrial membrane. However, low penetration in tumors greatly limits its application and efficacy. To develop a KLA-based cancer therapy, KLA-iRGD, a recombinant protein was constructed. It consists of the KLA peptide and iRGD (CRGDKGPDC), a tumor-homing peptide with high penetration into tumor tissue and cells. The conjugated KLA exhibits pro-apoptotic activity to prevent the growth of a tumor once it is inside the cell. Once KLA-iRGD is internalized in cultured tumor cells, via the activation of the receptor neuropilin-1, it spreads extensively throughout the mass of the tumor. The recombinant KLA-iRGD protein showed antitumor activity in vivo in mice and in vitro in tumor cell lines. Repeated treatment with KLA-iRGD greatly prevented tumor growth, resulting in a considerable reduction in tumor volume. According to our data, KLA-iRGD may serve as a potential anticancer agent with limited systemic toxicity and high selectivity for the treatment of MKN45 gastric cancer, which may lead to the enhancement of new targeted anticancer agents.

C0818, a novel curcumin derivative, interacts with Hsp90 and inhibits Hsp90 ATPase activity.

The aims of the present study were to estimate the affinity between 3,5-(E)-bis(3-methoxy-4-hydroxybenzal)-4-piperidinone hydrochloride (C0818) and heat shock protein 90 (Hsp90) and to investigate the inhibitory effects of this compound on Hsp90 ATPase activity. Fluorescence spectroscopy was used to examine the affinity between varying concentrations of C0818 and Hsp90, N-Hsp90, M-Hsp90 and C-Hsp90. Fluorescence intensities were recorded in the range of 290-510 nm at 293, 303 and 310 K, respectively. A colorimetric assay for inorganic phosphate (based on the formation of a phosphomolybdate complex and the subsequent reaction with malachite green) were used to examine the inhibitory effects of C0818 on Hsp90 ATPase activity. The equilibrium dissociation constant KD value of C0818 was found to be 23.412±0.943 µmol/L. The interaction between C0818 and Hsp90 was driven mainly by electrostatic interactions. C0818 showed the strongest affinity with C-Hsp90. These results conclusively demonstrate the inhibitory activity of C0818 on the activity of Hsp90 ATPase.

Structure Determinants of Lagunamide A for Anticancer Activity and Its Molecular Mechanism of Mitochondrial Apoptosis.

Marine natural products are served as attractive source of anticancer therapeutics, with the great success of "first-in-class" drugs, such as Yondelis, Halaven, and Brentuximab vendotin. Lagunamides A-C from marine cyanobacterium, Lyngbya majuscula, exhibit exquisite growth inhibitory activities against cancer cells. In this study, we have systematically investigated the structure-activity relationships (SARs) of a concise collection of lagunamide A and its analogues constructed by total chemical synthesis against a broad panel of cancer cells derived from various tissues or organs, including A549, HeLa, U2OS, HepG2, BEL-7404, BGC-823, HCT116, MCF-7, HL-60, and A375. The R configuration of lagunamide A at C-39 position was found to be the structure determinant for anticancer activity. Further molecular mechanism study in A549 cells revealed that lagunamide A induced caspase-mediated mitochondrial apoptosis. Accompanied with the dissipation of mitochondrial membrane potential (Δφm) and overproduction of reactive oxygen species (ROS), lagunamide A led to mitochondrial dysfunction and finally caused cell death. Moreover, both anti- and pro-apoptotic B-cell lymphoma 2 (Bcl-2) family proteins participated in lagunamide A-induced mitochondrial apoptosis, especially myeloid cell leukemia-1 (Mcl-1). Overexpression of Mcl-1 partly rescued A549 cells from lagunamide A-induced apoptosis. This study suggests that lagunamide A may exert anticancer property through mitochondrial apoptosis. Together, our findings would provide insightful information for the design of new anticancer drugs derived from lagunamides.

Quantitative proteomics reveals molecular mechanism of gamabufotalin and its potential inhibition on Hsp90 in lung cancer.

Gamabufotalin (CS-6) is a major bufadienolide of Chansu, which shows desirable metabolic stability and less adverse effect in cancer therapy. CS-6 treatment inhibited the proliferation of NSCLC in a nanomolar range. And CS-6 could induce G2/M cell cycle arrest and apoptosis in A549 cells. However, its molecular mechanism in antitumor activity remains poorly understood. We employed a quantitative proteomics approach to identify the potential cellular targets of CS-6, and found 38 possible target-related proteins. Among them, 31 proteins were closely related in the protein-protein interaction network. One of the regulatory nodes in key pathways was occupied by Hsp90. Molecular docking revealed that CS-6 interacted with the ATP-binding sites of Hsp90. In addition, CS-6 inhibited the chaperone function of Hsp90 and reduced expression of Hsp90-dependent client proteins. Moreover, CS-6 markedly down-regulated the protein level of Hsp90 in tumor tissues of the xenograft mice. Taken together, our results suggest that CS-6 might be a novel inhibitor of Hsp90, and the possible network associated with CS-6 target-related proteins was constructed, which provided experimental evidence for the preclinical value of using CS-6 as an effective antitumor agent in treatment of NSCLC.

Secondary metabolites of Xylaria sp., an endophytic fungus from Taxus mairei.

One new metabolite 3,7-dimethyl-9-(-2,2,5,5-tetramethyl-1,3-dioxolan-4-yl)nona-1,6-dien-3-ol, together with nine known compounds, were isolated from the strain Xylaria sp., an endophytic fungus of Taxus mairei. Their structures were deduced from 1D and 2D NMR data. In vitro cytotoxicity and antibacterial activity of these compounds were evaluated. Some of them exhibited substantial activity.

CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients.

Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells, and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn't affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies.