Treating tumor-bearing mice with vitamin D3 diminishes tumor-induced myelopoiesis and associated immunosuppression,and reduces tumor metastasis and recurrence

Metastatic Lewis lung carcinoma (LLC-LN7) tumors that secrete granulocyte/macrophage-colonystimulating factor (GM-CSF) stimulate myelopoiesis and induce bone marrow-derived immunosuppressor cells that are homologous to granulocyte/macrophage progenitor cells. In vitro treatment of the LLC-LN7 cells with 1,25-dihydroxyvitamin D₃ reduced tumor cell production of suppressor-inducing activity, although suppressor-inducing activity could be restored by reconstituting the tumor supernatants with recombinant GM-CSF. Treatment of mice having LLC-LN7 tumors with vitamin D₃ reduced tumor production of GM-CSF and the frequency of myeloid progenitor cells. This was associated with a reduction in immunosuppressor activity and an increase in T cell function. Vitamin D₃ treatment of mice having palpable tumors transiently retarded tumor growth, but caused a prominent reduction in tumor metastasis. Treating mice with vitamin D₃ after tumor excision resulted in a reduction in the tumor-induced myelopoietic stimulation and associated immunosuppressive activity, and enhanced T cell function. These mice had a markedly reduced incidence of tumor recurrence. The results of this study suggest that vitamin D₃ treatment of mice with GM-CSF-secreting tumors can interrupt the myelopoiesis-associated immunosuppressor cascade and, in turn, reduce tumor metastasis and recurrence.This study was supported in part by grants from the Medical Research Service of the Department of Veterans Affairs and by grants CA-45080 and CA-48080 from the National Institutes of Health     

Squamous cell carcinoma of the lip

We reviewed 117 consecutive patients with squamous cell carcinoma of the lip. The retrospective review includes age, race, location, risk factors, TNM classification, histologic differentiation, treatment methods, recurrent disease, site of recurrence, and follow-up status. Results reveal prognosis is related to original tumor size, location, local recurrence, histologic grade, and presence of cervical metastasis. The presence of cervical lymph node disease reduces the survival from 90 to 50 percent; the survival after recurrent disease to the neck is 10 percent. When a prophylactic suprahyoid neck dissection shows involvement with tumor, 83 percent of patients have metastasis to cervical lymph nodes. The overall recurrence rate is 20 percent. Over 60 percent of the recurrent disease is due to tumors less than 4 cm in diameter. The local recurrence rate is 7 percent, but reexcision of the local recurrence gives a 75 percent cure rate. Aggressive surgical treatment is recommended for identifiably poor prognostic lesions and includes surgical excision, prophylactic suprahyoid neck dissection, and possible radical neck dissection.     

门静脉高压脾功能亢进对大鼠肝癌发生率的影响

目的:通过对门静脉高压脾功能亢进大鼠药物诱导肝癌过程中进行脾脏切除,探讨门静脉高压脾功能亢进对大鼠肝癌发生率的影响。方法:将雌雄SD大鼠性别内分别分为对照组、脾亢组、脾亢切脾组,脾功能亢进大鼠模型采用门静脉缩窄术联合脾静脉结扎术进行制备,各组均予以DEN(二乙基亚硝胺)腹腔注射,按体重20mg/kg给药,每周3次,12周停药,14周处死。其中,脾亢脾切除组于给药第四周进行脾切除术,手术恢复期间持续给药。观察各组实验动物的肝脏大体变化及病理改变,计算成瘤率。结果:实际成瘤率显示脾亢组较对照组明显升高,而雄性脾亢切脾组的成瘤率较脾亢组有所降低。雌性脾亢切脾组成瘤率同脾亢组差异不明显。结论:门静脉高压脾功能亢进状态下进行脾切除,对于雄性能减低肝癌发生的风险,对于雌性的意义不大,给临床实际工作提供了新的思路。     

Immune reconstitution prevents metastatic recurrence of murine osteosarcoma

Primary tumors developing in immunocompetent hosts escape immunosurveillance by acquiring immune evasive properties. This raises the prospect that metastases derived from such tumors will also evade immunity. To investigate whether immune surveillance plays a role in preventing metastases, we studied a murine model which mimics the clinical progression of osteosarcoma: primary tumor growth in the lower extremity, amputation, minimal residual disease followed by the development of overt metastases. K7M2 implants readily escaped immune surveillance since normal BALB/c mice, T cell deficient SCID and T/NK cell deficient SCID-bg mice showed no difference in the rate of growth of primary osteosarcomas. However, both SCID and SCID-bg mice had higher rates of metastases than immunocompetent mice. Similarly, immune reconstitution following transfer of naive T cells to SCID or SCID-bg mice did not impact primary tumor growth, but significantly diminished metastatic recurrence. T cells in osteosarcoma bearing mice produced IFNγ in response to tumor and IFNγ production by immune reconstituting T cells was required to prevent metastases. These results demonstrate an important role for T cell based immune surveillance in preventing metastases, even when metastases develop from tumors that adeptly evade immunosurveillance. The results further suggest that T cell depleting cancer therapies may eliminate beneficial immune responses and that immune reconstitution of lymphopenic cancer patients could prevent metastatic recurrence of solid tumors. By acceptance of this article, the publisher or recipient acknowledges right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Animal care was provided in accordance with procedures outlined in the “Guide for the Care and Use of Laboratory Animals” (NIH Pub. No. 86-23, 1996). This project was funded in whole or part with funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-56000.     

How well is the probability of tumor cure after fractionated irradiation described by Poisson statistics?

The probability of tumor cure in a homogeneous population of tumors exposed to fractionated radiotherapy was modeled using numerical simulations and compared with the predictions of Poisson statistics, assuming exact knowledge of the relevant tumor parameters (clonogen number, radiosensitivity, and growth kinetics). The results show that although Poisson statistics (based on exact knowledge of all parameters) accurately describes the probability of tumor cure when no proliferation occurs during treatment, it underestimates the cure rate when proliferation does occur. In practice, however, the inaccuracy is not likely to be more than about 10%. When the tumor parameters are unknown and are estimated by fitting an empirical Poisson model to tumor-cure data from a homogeneous population of proliferative tumors, the resulting estimates of tumor growth rate and radiosensitivity accurately reflect the true values, but the estimate of initial clonogen number is biased downward. A new formula that is more accurate than Poisson statistics in predicting the probability of tumor cure when proliferation occurs during treatment is discussed.     

Transient activation of tumor-associated T-effector/memory cells promotes tumor eradication via NK-cell recruitment: minimal role for long-term T-cell immunity in cure of metastatic disease

Local delivery of IL-12 and GM-CSF to advanced primary tumors results in T- and NK-cell-dependent cure of disseminated disease in a murine spontaneous lung metastasis model. Post-therapy functional dynamics of cytotoxic T- and NK-cells were analyzed in primary and metastatic tumors to determine the specific roles of each subset in tumor eradication. Time-dependent depletion of CD8+ T and NK-cells demonstrated that CD8+ T-cells were critical to eradication of metastatic tumors within 3 days of treatment, but not later. In contrast, NK-cells were found to be essential to tumor regression for at least 10 days after cytokine delivery. Analysis of tumor-infiltrating lymphocyte populations in post-therapy primary tumors demonstrated that treatment resulted in the activation of tumor-associated CD8+ T-cells within 24 h as determined by IFNγ and perforin production. T-cell activity peaked between days 1 and 3 and subsided rapidly thereafter. Activation was not accompanied with an increase in cell numbers suggesting that treatment mobilized pre-existing T-effector/memory cells without inducing proliferation. In contrast, therapy resulted in a ≥3-fold enhancement of both the quantity and the cytotoxic activity of NK-cells in primary and metastatic tumors on day 3 post-therapy. NK-cell activity was also transient and subsided to pre-therapy levels by day 5. Depletion of CD4+ and CD8+ T-cells prior to treatment completely abrogated NK-cell infiltration into primary and metastatic tumors demonstrating the strict dependence of NK-cell recruitment on pre-existing T-effector/memory cells. Treatment failed to induce significant NK-cell infiltration in IFNγ-knockout mice establishing the central role of IFNγ in NK-cell chemotaxis to tumors. These data show that transient activation of tumor-associated T-effector/memory and NK-cells, but not long-term CD8+ T-cell responses, are critical to suppression of metastatic disease in this model; and reveal a novel role for pre-existing adaptive T-cell immunity in the recruitment of innate effectors to tumors. This work was supported by NIH/NCI grant R01-CA100656-01A1 to N.K.E.     

微波高温灭活及自体髂骨异体骨粒复合骨水泥修复治疗长骨骨巨细胞瘤骨缺损

目的：探讨微波高温灭活及自体髂骨、异体骨粒复合骨水泥修复骨巨细胞瘤病灶刮除后骨缺损的临床应用效果。方法：应用原位分离插入式微波天线高温灭活技术,自体髂骨、异体骨粒复合骨水泥修复21例长骨骨巨细胞瘤术后骨缺损,从手术技术、肿瘤复发情况、肢体关节功能等方面全面综合评价此方法临床应用效果。结果：21例患者均获得骨性愈合,无骨折及内固定断裂发生,2例复发,复发率9.8%;肢体关节功能优18例（85.7%）、良3例（14.3%）、中差0例。结论：微波高温能彻底杀灭肿瘤组织降低复发率,自体髂骨保证与近关节软骨下骨愈合,异体骨粒复合骨水泥能良好充填残余瘤腔、且具有良好的生物力学性能,以防发生关节软骨面塌陷。     

涉及桡骨远端骨巨细胞瘤的外科手术疗效比较

目的：探讨和比较不同手术术式治疗涉及桡骨远端的骨肉瘤的手术适应症选择,临床疗效和安全性。方法：将2005 年-2014 年我院收治的涉及到桡骨远端并进行外科手术治疗的骨巨细胞瘤患者共88 例进行回顾性分析。根据影像学Campanacci分级,主 要手术方法分为以下三种,分别为：A组：微波天线高温原位灭活,自体髂骨,异体骨粒符合骨水泥重建修复术;B：瘤骨切除并腓 骨移植术;C：瘤骨刮除灭活并原位植骨术。结合详实的随访资料对两组患者在术后的复发率,腕关节功能（Enneking）等情况给予 分析和评价。结果：A 组复发率为10.87 %,腕关节功能MSTS93 功能评分为26.32± 2.92分。B 组复发率为0,腕关节术后的 MSTS93 功能评分为22.85± 4.16 分。C 组复发率为30.24 %。腕关节术后的MSTS93 功能评分为26.97± 2.84 分。三组相比,A、 B 与C 组在复发率中有明显统计学差异（P<0.05）,A 组、C组的术后功能评分明显优于B 组（P<0.05）,但两组之间无统计学差异 （P>0.05）。A 组中有1 例切口表层感染,B 组中有2 例皮肤感染,均经加强换药后治愈。结论：瘤段切除手术能够有效的降低复发 率,但局部功能恢复较差,容易出现切口感染等并发症。微波灭活手术可以有效的杀灭肿瘤组织并保证良好的功能性,但复发率 相对较高。在临床工作中应根据患者具体病情和需要给予针对性的手术方案。     

Biological properties of human melanoma cells in culture

Summary Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary. THis work was supported in part by USPHS Grant No. 5 T01 AI00332-06 from the National Institutes of Health, Contract E73-2001-N01-CP-3-3237 from the Virus Cancer Program of the National Cancer Institute, and USPHS Grant No. 0H00714-02 from the National Institute for Occupational Safety and Health.     

Electrochemical treatment of mouse and rat fibrosarcomas with direct current

Electrochemical treatment (ECT) of cancer utilizes direct current to produce chemical changes in tumors. ECT has been suggested as an effective alternative local cancer therapy. However, a methodology is not established, and mechanisms are not well studied. In vivo studies were conducted to evaluate the effectiveness of ECT on animal tumor models. Radiation-induced fibrosarcomas were implanted subcutaneously in 157 female C3H/HeJ mice. Larger rat fibrosarcomas were implanted on 34 female Fisher 344 rats. When the spheroidal tumors reached 10 mm in the mice, two to five platinum electrodes were inserted into the tumors at various spacings and orientations. Ten rats in a pilot group were treated when their ellipsoidal tumors were about 25 mm long; electrode insertion was similar to the later part of the mouse study, i.e., two at the base and two at the center. A second group of 24 rats was treated with six or seven electrodes when their tumors were about 20 mm long; all electrodes were inserted at the tumor base. Of the 24 rats, 12 of these were treated once, 10 were treated twice, and 2 were treated thrice. All treated tumors showed necrosis and regression for both mice and rats; however, later tumor recurrence reduced long-term survival. When multiple treatments were implemented, the best 3 month mouse tumor cure rate was 59.3%, and the best 6 month rat tumor cure rate was 75.0%. These preliminary results indicate that ECT is effective on the radiation-induced fibrosarcoma (RIF-1) mouse tumor and rat fibrosarcoma. The effectiveness is dependent on electrode placement and dosage. Bioelectromagnetics 18:14–24, 1997. © 1997 Wiley-Liss, Inc.     

DC immunotherapy is highly effective for the inhibition of tumor metastasis or recurrence,although it is not efficient for the eradication of established solid tumors

Dendritic cell (DC)-based immunotherapy has not been as effective as expected in most solid tumors even in the murine model, particularly in renal cell carcinoma (RCC). Our investigation was initiated to identify what causes the limitations of DC-based immunotherapy in solid RCC. We have investigated immunosuppressive factors from tumors and their effects on DC migration, as well as cytotoxic T lymphocyte (CTL) response and lymphocyte infiltration into the tumor mass upon vaccination with mouse renal adenocarcinoma (Renca) cell lysate-pulsed bone marrow (Bm)-derived DC in tumor-bearing mice. We also investigated pulmonary metastasis- and tumor recurrence-inhibitory effects of DC-vaccination in the solid tumor-bearing mice. In these experiments, we found that the limitations of DC-based immunotherapy to solid RCC likely result from tumor-mediated TGF-β hindrance of immune attack rather than insufficient immune induction by DC therapy. In fact, the CTL response induced by DC therapy was quite sufficient and functional for the inhibition of tumor recurrence after surgery or of tumor metastasis induced by additional tumor-challenge to the tumor-bearing mice. Taken together, our present results obtained in mouse model suggest the potential of DC immunotherapy in tumor patients for hindering or blocking disease progression by inhibition of tumor metastasis and/or tumor recurrence after surgery.     

Toward a Long-Lasting Immune Prevention of HER2 Mammary Carcinomas: Directions from Transgenic Mice

Several theoretical and practical issues differentiate immune prevention from tumor immune therapy. The latter seeks to induce a rapid reaction against a life-threatening tumor, whereas prevention is dictated by the need to maintain constant surveillance of a situation in which an event is foreseen, but may not occur. The time frame of successful prevention is therefore long and often lifelong. Time itself is thus a key factor in the elaboration of vaccines to prevent tumor growth and its great length in preventive management poses new immunological problems that cannot be studied in short-term vaccination-challenge experiments. Many recent data indicate that HER2 receptor displays several features of an ideal tumor associated antigen and that an immune response can significantly alter HER2 tumor progression. We are thus using vaccination in the immune prevention and cure of carcinomas in HER2 transgenic mice in the search for a rationale for the application of preventive and curative vaccination for patients with HER2/ErbB-2 neoplastic lesions or at risk of recurrence after successful surgery. The design of effective immunopreventive approaches that can be translated to human situations is an important issue. A molecularly defined, effective and validated anti HER2 vaccine and the definition of immune mechanisms leading to the inhibition of HER2-driven neoplastic proliferation may provide a new way of treating these patients.     

The therapeutic effects of an immunopotentiator,PS-K,on 3-methylcholanthrene-induced autochthonous tumors in C57BL/6 mice in combination with the surgical removal of primary sites

Summary We investigated the therapeutic effects of an immunopotentiator PS-K on recurrent or metastatic tumors observed after the surgical removal of MCA-induced primary tumors in autochthonous C57BL/6 mice and on the survival time of treated mice. The MST of mice treated with PS-K at various times (59.8 63.4 days) was prolonged as compared with that of mice treated by surgery alone (48.6 days). Local recurrence of tumors was found in 36 of 66 mice (54.6%) treated by surgery alone, whereas it was inhibited significantly (P<0.05) when treatment with PS-K was started 1 day after the surgery and occurred in 22 of 64 mice (34.4%) when PS-K was given for 5 days in 1 week, or in 22 of 66 mice (33.3%) when PS-K was administered twice a week for 7 weeks. The MSTs of mice with local recurrence were also found to be prolonged as compared with those of mice treated by surgery alone (54.8 67.5 days vs 49.8 days). The MSTs of mice without tumor recurrence were also prolonged significantly (P<0.05 0.001) by combinations of PS-K at various times, although most of the mice died of metastatic tumors even in the groups of mice where a combined treatment with PS-K had been administered. The above findings suggest that the administration of PS-K inhibits the growth of recurrent or metastatic tumor cells in autochthonous mice after the surgical removal of the primary tumors.This work was supported in part by Grants in Aid for Cancer Research from the Japanese Ministries of Education, Science and Culture and of Health and Welfare Abbreviations used: MCA; 3-methylcholanthrene, CY; cyclophosphamide, MST; mean survival time     

Comparison of Immunity in Mice Cured of Primary/Metastatic Growth of EMT6 or 4THM Breast Cancer by Chemotherapy or Immunotherapy

Purpose

We have compared cure from local/metastatic tumor growth in BALB/c mice receiving EMT6 or the poorly immunogenic, highly metastatic 4THM, breast cancer cells following manipulation of immunosuppressive CD200:CD200R interactions or conventional chemotherapy.

Methods

We reported previously that EMT6 tumors are cured in CD200R1KO mice following surgical resection and immunization with irradiated EMT6 cells and CpG oligodeoxynucleotide (CpG), while wild-type (WT) animals developed pulmonary and liver metastases within 30 days of surgery. We report growth and metastasis of both EMT6 and a highly metastatic 4THM tumor in WT mice receiving iv infusions of Fab anti-CD200R1 along with CpG/tumor cell immunization. Metastasis was followed both macroscopically (lung/liver nodules) and microscopically by cloning tumor cells at limiting dilution in vitro from draining lymph nodes (DLN) harvested at surgery. We compared these results with local/metastatic tumor growth in mice receiving 4 courses of combination treatment with anti-VEGF and paclitaxel.

Results

In WT mice receiving Fab anti-CD200R, no tumor cells are detectable following immunotherapy, and CD4+ cells produced increased TNFα/IL-2/IFNγ on stimulation with EMT6 in vitro. No long-term cure was seen following surgery/immunotherapy of 4THM, with both microscopic (tumors in DLN at limiting dilution) and macroscopic metastases present within 14 d of surgery. Chemotherapy attenuated growth/metastases in 4THM tumor-bearers and produced a decline in lung/liver metastases, with no detectable DLN metastases in EMT6 tumor-bearing mice-these latter mice nevertheless showed no significantly increased cytokine production after restimulation with EMT6 in vitro. EMT6 mice receiving immunotherapy were resistant to subsequent re-challenge with EMT6 tumor cells, but not those receiving curative chemotherapy. Anti-CD4 treatment caused tumor recurrence after immunotherapy, but produced no apparent effect in either EMT6 or 4THM tumor bearers after chemotherapy treatment.

Conclusion

Immunotherapy, but not chemotherapy, enhances CD4⁺ immunity and affords long-term control of breast cancer growth and resistance to new tumor foci.     

Anti-tumor immune responses following neoadjuvant immunotherapy with a recombinant adenovirus expressing HSP72 to rodent tumors

Gene modification of tumor cells is commonly utilized in various strategies of immunotherapy preventive both as treatment and a means to modify tumor growth. Gene transfer prior to surgery as neoadjuvant therapy has not been studied systematically. We addressed, whether direct intra-tumoral injection of a recombinant adenovirus expressing the immunomodulatory molecule, heat shock protein 72 (ADHSP72), administered prior to surgery could result in sustainable anti-tumor immune responses capable of affecting tumor progression and survival in a number of different murine and rat tumor models. Using intra-dermal murine models of melanoma (B16), colorectal carcinoma (CT26), prostate cancer (TrampC2) and a rat model of glioblastoma (9L), tumors were treated with vehicle or GFP expressing adenovirus (ADGFP) or ADHSP72. Tumors were surgically excised after 72 h. Approximately 25–50% of animals in the ADHSP72 treatment group but not in control groups showed sustained resistance to subsequent tumor challenge. Tumor resistance was associated with development of anti-tumor cellular immune responses. Efficacy of ADHSP72 as neoadjuvant therapy was dependent on the size of the initial tumor with greater likelihood of immune response generation and tumor resistance associated with smaller tumor size at initial treatment. ADHSP72 neoadjuvant therapy resulted in prolonged survival of animals upon re-challenge with autologous tumor cells compared to ADGFP or vehicle control groups. To study the effects on tumor progression of distant metastases, a single tumor focus of animals with multifocal intra-dermal tumors was treated. ADHSP72 diminished progression of the secondary tumor focus and prolonged survival, but only when the secondary tumor focus was <50 mm³ . Our results indicate that gene modification of tumors prior to surgical intervention may be beneficial to prevent recurrence in specific circumstances.     

Differential responsiveness of various substrains of inbred strain 2 guinea pigs to immunotherapy with the methanol extraction residue (MER) of BCG

Summary The immunotherapeutic effects of the methanol extraction residue (MER) of BCG were investigated in strain 2 guinea pigs bearing the transplantable line 10 hepatocarcinoma, a tumor originally induced in guinea pigs at the National Institutes of Health (NIH) by ingestion of the carcinogen diethylnitrosamine. MER was more effective in mediating tumor regression in guinea pigs obtained from the Weizmann Institute of Science (WI), Rehovot, Israel, than in animals obtained from the National Institutes of Health (NIH). These differences indicate the dramatic effects which minor histoincompatibilities between cancer cells and animal substrains may have on experimental results, and highlight the need for immunotherapy experiments to be conducted on laboratory tumors grown in their autochthonous hosts. MER was effective only when injected directly into growing tumor nodules and had no effect on tumor development when administered distally. In contrast, all animals which received both MER and tumor cells developed specific cell-mediated anti-tumor immune responsiveness at higher levels than did non-MER-treated tumor-bearing controls as measured by delayed cutaneous hypersensitivity and in vitro lymphocyte reactivity experiments. Furthermore, the results of the latter but not the former studies suggested that guinea pigs which received MER were able to mount such an immune response more rapidly than their non-treated counterparts. This apparent stimulation of anti-tumor immunity was observed in treated animals regardless of substrain or site of MER injection, and could not be correlated with the outcome of immunotherapy.     

Prediction of recurrence in giant cell bone tumors by DNA cytometry.

The most interesting therapeutic aspect of giant cell bone tumors is which patients can be cured without a risk of recurrence by intralesional surgery (curettage). To find out the suitability of some DNA cytometric and morphometric parameters for showing differences between this group of patients (n = 9) and those with recurrence (n = 12), the parameters mean ploidy, 2cDI (mean square deviation of the tumor cell DNA content from the normal 2c value), mean nuclear area and its variability were calculated from cytologic specimens prepared by a cell separation technique from formalin-fixed, paraffin-embedded tissues, measuring the values of 100 stromal cells per case by a TV image analysis system. Further measurements were performed on 19 cases of different diseases of the bone and on an additional 17 cases of giant cell tumors without follow-up. The 2cDI allowed us to distinguish the two groups of patients, with and without recurrence, without overlap; even the lowest value for patients with recurrence was higher than the highest value for cured ones. Mean ploidy analysis resulted in a less convincing discrimination of the patients. Mean nuclear area and its variability failed to predict recurrence. Single-cell DNA cytometry provided a parameter, 2cDI, that was able to predict recurrence in patients with giant cell bone tumors with high sensitivity.     

Monitoring HOTTIP levels on extracellular vesicles for predicting recurrence in surgical non-small cell lung cancer patients

In resected non-small cell lung cancer (NSCLC), postsurgical recurrence is the major factor affecting long-term survival. The identification of biomarkers in extracellular vesicles (EV) obtained from serial blood samples after surgery could enhance early detection of relapse and improve NSCLC outcome. Since EV cargo contains long non-coding RNAs (lncRNAs), we aimed to analyze whether the oncogenic lncRNA HOTTIP, which higher expression in tumor tissue was related to worse outcome in NSCLC, could be detected in EV from NSCLC patients and serve as recurrence biomarker. After purification of EVs by ultracentrifugation in 52 serial samples from 18 NSCLC patients, RNA was isolated and HOTTIP was quantified by Real time PCR. We observed that patients that relapsed after surgery displayed increased postsurgical EV HOTTIP levels in comparison with presurgical levels. In the relapsed patients with several samples available between surgery and relapse, we observed an increment in the EV HOTTIP levels when approaching to relapse, which indicated its potential utility for monitoring disease recurrence. When we focused in EV HOTTIP levels in the first post-surgical sample, we observed that the detection of an increment of the expression levels in comparison to presurgical sample, predicted recurrence with high sensitivity (85.7%) and specificity (90.9%) and that patients had shorter time to relapse and shorter overall survival. In conclusion, our pilot study showed that EV HOTTIP is a potential biomarker for monitoring disease recurrence after surgery in NSCLC.     

Cbl induced ubiquitination of HER2 mediate immune escape from HER2-targeted CAR-T

Breast cancer (BC) with high HER2 expression has higher recurrence rate and worse prognosis, and its immunotherapy is promising. Based on the high expression of HER2, develop Chimeric Antigen Receptor T-cell (CAR-T) and PDL-1 immunotherapy, and study the molecular pathways of related immune cells and recurrence. HER2-CAR-T cells were constructed using retroviruses, and their specific recognition and immune effects on HER2+ BC cells were verified by in vivo and in vitro experiments. PDL-1 was used as adjuvant immunotherapy, knocking down PDL-1 in tumor cells or dendritic cells, or depleted macrophages to study immune pathways. The negative regulation of HER2 by cbl was determined by IP, ubiquitination experiments, and segmented plasmids, elucidating the molecular mechanism of HER2+ BC recurrence after immunotherapy. HER2-CAR-T specifically recognizes HER2-positive tumor cells and inhibits tumor growth in vivo and in vitro, and anti-PDL1 treatment enhances the therapeutic effect of HER2-CAR-T on tumors. HER2-CART therapy eradicated solid tumors after PDL1 knockdown in dendritic cells. Immunotherapy of relapsed tumors lost HER2 expression by upregulating cbl. HER2-CAR-T shows specific recognition of HER2+ cells and can mediate immune response therapy with the cooperation of PDL-1.     

Requirement of hydrocortisone and insulin for extended proliferation and passage of rat keratinocytes

Summary A procedure for the preparation and cultivation of rat epidermal basal cells from full thickness skin resulted in greater than 99% viability and 90% plating efficiency. However, attempts to subculture monolayers of these epithelial cells grown in medium with serum as the only supplement were totally unsuccessful. When hydrocortisone and insulin were added to the medium, subcultivation of primary growth was obtained. It was demonstrated that hydrocortisone at concentrations as low as 0.1 μg/ml was necessary for at least the initial attachment of the cells to the substrate—an essential step in subcultivation. Increasing concentrations of insulin (0.1 to 50 μg/ml) caused the rate of proliferation and the cell density to increase, but insulin alone did not support subcultivation. This work was supported by Grants 1-R01-CA-19988 and 5-R01-AM-15206 from the National Institutes of Health, U.S. Public Health Service.