Tumor-derived microparticles induce bone marrow-derived cell mobilization and tumor homing: a process regulated by osteopontin.

Acute chemotherapy can induce rapid bone-marrow derived pro-angiogenic cell (BMDC) mobilization and tumor homing, contributing to tumor regrowth. To study the contribution of tumor cells to tumor regrowth following therapy, we focused on tumor-derived microparticles (TMPs). EMT/6 murine-mammary carcinoma cells exposed to paclitaxel chemotherapy exhibited an increased number of TMPs and significantly altered their angiogenic properties. Similarly, breast cancer patients had increased levels of plasma MUC-1(+) TMPs following chemotherapy. In addition, TMPs from cells exposed to paclitaxel induced higher BMDC mobilization and colonization, but had no increased effect on angiogenesis in Matrigel plugs and tumors than TMPs from untreated cells. Since TMPs abundantly express osteopontin, a protein known to participate in BMDC trafficking, the impact of osteopontin-depleted TMPs on BMDC mobilization, colonization, and tumor angiogenesis was examined. Although EMT/6 tumors grown in mice inoculated with osteopontin-depleted TMPs had lower numbers of BMDC infiltration and microvessel density when compared with EMT/6 tumors grown in mice inoculated with wild-type TMPs, no significant difference in tumor growth was seen between the two groups. However, when BMDCs from paclitaxel-treated mice were injected into wild-type EMT/6-bearing mice, a substantial increase in tumor growth and BMDC infiltration was detected compared to osteopontin-depleted EMT/6-bearing mice injected with BMDCs from paclitaxel-treated mice. Collectively, our results suggest that osteopontin expressed by TMPs play an important role in BMDC mobilization and colonization of tumors, but is not sufficient to enhance the angiogenic activity in tumors.

Circulating blood extracellular vesicles as a tool to assess endothelial injury and chemotherapy toxicity in adjuvant cancer patients.

Extracellular vesicles (EVs) are subcellular membrane blebs that include exosomes and microparticles, which represent a potential source for cancer biomarker discovery. We assess EVs characteristics as a tool to evaluate the endothelial and anti-tumor treatment injury during adjuvant chemotherapy in breast (BC) and colon cancer (CC) patients. Blood samples were taken from 29 BC and 25 CC patients before and after chemotherapy, as well as from healthy control donors (HC). Circulating blood EVs were isolated and characterized by size/concentration, membrane antigens for cell origin, thrombogenicity, and protein content. We observed higher EVs concentration and particle size in CC patients after chemotherapy compared with HC. Higher levels of endothelial EVs (CD144-positive) and vascular endothelial growth factor receptor 1 (VEGFR1), apparently as an indication of endothelial dysfunction, were found in all cancer patients, regardless of a given treatment, compared to HC. Levels of EVs labeled CD62E, CD34+41-, the lymphocyte markers CD11+ and CD-14+, Annexin-V, and the coagulation proteins TF and TFPI, however, sometimes demonstrate significant differences between patients, although HC did not show significant differences between patients pre- and post-chemotherapy. Most importantly, increasing levels of EVs encapsulated Angiostatin were found in patients with CC, while chemotherapy treatment leads to its notable rise in circulating blood EVs. Our results demonstrate the potential of EVs encapsulated Angiostatin as a tool to evaluate endothelial damage during adjuvant chemotherapy in BC and CC patients.

Breast Cancer-Derived Microparticles Reduce Cancer Cell Adhesion, an Effect Augmented by Chemotherapy.

Tumor cell heterogeneity is primarily dictated by mutational changes, sometimes leading to clones that undergo a metastatic switch. However, little is known about tumor heterogeneity following chemotherapy perturbation. Here we studied the possible involvement of tumor-derived extracellular vesicles, often referred to as tumor-derived microparticles (TMPs), as mediators of the metastatic switch in the tumor microenvironment by hindering cell adhesion properties. Specifically, we show that highly metastatic or chemotherapy-treated breast cancer cells shed an increased number of TMPs compared to their respective controls. We found that these TMPs substantially reduce cell adhesion and disrupt actin filament structure, therefore increasing their biomechanical force pace, further implicating tumor cell dissemination as part of the metastatic cascade. Our results demonstrate that these pro-metastatic effects are mediated in part by CD44 which is highly expressed in TMPs obtained from highly metastatic cells or cells exposed to chemotherapy when compared to cells with low metastatic potential. Consequently, when we inhibited CD44 expression on TMPs by a pharmacological or a genetic approach, increased tumor cell adhesion and re-organized actin filament structure were observed. We also demonstrated that breast cancer patients treated with paclitaxel chemotherapy exhibited increased CD44-expressing TMPs. Overall, our study provides further insights into the role of TMPs in promoting metastasis, an effect which is augmented when tumor cells are exposed to chemotherapy.

Effect of early administration of the N-methyl-d-aspartate receptor antagonist amantadine on the development of postmastectomy pain syndrome: a prospective pilot study.

UNLABELLED: Postmastectomy pain syndrome (PMPS) is a neuropathic pain syndrome that might develop after breast surgery. Like many other forms of neuropathic pain, it is relatively resistant to treatment and negatively affects the quality of life. A double-blind, randomized, placebo-controlled pilot trial was conducted to study the analgesic efficacy of perioperative administration of the N-methyl-D-aspatrate (NMDA) receptor antagonist amantadine in preventing PMPS after mastectomy plus axillary lymph node dissection (ALND). In the study group, a regimen of 200 mg/day of amantadine was started 1 day before surgery and continued for 14 days, whereas the control group received a placebo. Patients were required to indicate the exact location of their pain and to record its level at 1, 3, and 6 months after surgery. Neurologic examination and Quantitative Thermal Testing (QTT) were performed 1 and 6 months after surgery. On both the neurologic examination and the QTT, all patients, regardless of the perioperative intervention (amantadine or placebo), presented evidence for nerve injury, manifested primarily by painful hypoesthesia (anesthesia dolorosa) in the axilla or inner arm. PMPS persisted for the entire duration of the study in 82% of the patients who were available for follow-up. The average intensity of the pain was moderate in both groups and tended not to decline over time. No differences between the 2 groups in any of the outcome parameters reached statistical significance. According to the results of the present pilot study, the NMDA antagonist amantadine does not prevent the development of PMPS in patients who undergo breast surgery with ALND. PERSPECTIVE: Breast surgery that involves ALND seems to uniformly cause nerve injury, which cannot be prevented by the perioperative administration of 200 mg of amantadine. It is most commonly presented by painful hypoesthesia or anesthesia dolorosa in the axillary/inner arm area, which is moderate in intensity and likely to persist for at least 6 months.

Chemotherapy administration to breast cancer patients affects extracellular vesicles thrombogenicity and function.

Breast cancer (BC) is the most prevalent type of malignancy in women. Extracellular vesicles (EVs) are subcellular membrane blebs that include exosomes and microparticles. STUDY AIMS: To elucidate the effects of chemotherapy administration on BC patients' EVs characteristics and their effects on endothelial cells (EC) functions. METHODS: EVs were isolated from the blood samples of 54 BC patients treated by chemotherapy (25 neo-adjuvant, 29 adjuvant) and from 20 healthy women (control group). Blood samples were taken before chemotherapy and on the day of last chemotherapy administration. In some patients, samples were also evaluated 24 hours after chemotherapy treatment. EVs were characterized by cell origin, thrombogenicity and cytokine content. EVs effects on coagulation, migration, apoptosis and proliferation of endothelial cells were assessed as well. RESULTS: Patient characteristics of the two subgroups were similar except for tumor size. Change in EV expression of BC markers, MUC1 and EpCAM, were found in patient subgroups. EC-EVs were significantly higher in both patient subgroups compared to healthy controls. Higher EVs pro-coagulant activity was found at the end of chemotherapy and a significant increase in the ratio between tissue factor (TF) and TF pathway inhibitor was documented after the first 24hours of exposure to doxorubicin treatment. Furthermore, EVs of neo-adjuvant patients obtained before chemotherapy contained more pro-angiogenic proteins, reduced endothelial cells apoptosis and increased their migration compared to EVs obtained at the same timing from adjuvant patients. CONCLUSIONS: EVs may serve as a biomarker for chemotherapy-related thrombogenicity and may indicate vascular damage even before chemotherapy.

Sister Mary Joseph's nodule as a presenting sign of internal malignancy.

CASE 1: A 64-year-old, otherwise healthy woman was referred to the surgery clinic for a presumed umbilical hernia. On physical examination, a cutaneous nodule was noted on the umbilical region and the patient was referred to the dermatology clinic. The patient was reexamined and an erythematous nodule was observed in the umbilicus measuring 2.5 cm in diameter. The patient denied pain, change in bowel habits, or weight loss. There were no other abdominal masses, no sign of ascites, and no regional lymphadenopathy. A skin biopsy from the nodule showed mucinous adenocarcinoma. Immunohistochemical staining was positive for carcinoembryonic antigen, and negative for cytokeratin (CK)7 and CK20. These results were consistent with a Sister Mary Joseph's nodule and led to the diagnosis of an occult colon carcinoma. The patient had no risk factors for colorectal carcinoma. The patient underwent surgery in another hospital, and died 3 months after the initial diagnosis of Sister Mary Joseph's nodule. CASE 2: A 73-year-old woman was referred to the dermatology clinic for evaluation of a painful, ulcerated, 3-cm lesion in the umbilicus (Figure 1). She was otherwise asymptomatic. A skin biopsy showed neoplastic glandular cells infiltrating among collagen bundles (Figure 2). Stainings for mucin and for CK7 were positive, while staining for CK20 was negative. An abdominopelvic CT scan demonstrated a 3.5-cm space-occupying lesion in the liver. Results of gastroscopy, colonoscopy, chest computed tomographic (CT) scan, and mammography were normal. Serum levels of the tumor-associated protein CA125 were elevated to 164 units, while those of CA 19-9 and carcinoembryonic antigen were within normal range. A gynecologic examination and a transvaginal ultrasound were normal. The patient had no personal or family history of any malignancy or any risk factors for developing a carcinoma. The patient was scheduled for a palliative resection of the umbilical nodule, combined with a laparoscopic inspection in search of the undetected primary tumor. She refused surgery and was lost to follow-up. She died 4 months after the initial diagnosis of umbilical metastasis. CASE 3: A 51-year-old man was aware of a silent mass in his umbilicus for 2 years without seeking medical advice. Following 2 weeks of increasing pain in this area, he was referred to the emergency room for a suspected incarcerated umbilical hernia. Surgery revealed a mass attached to the fascia and peritoneal fat. The mass was removed and diagnosed as a poorly differentiated adenocarcinoma, staining positively for carcinoembryonic antigen, and negatively for CK20, CK7, prostate-specific antigen, and prostatic acid phosphatase. Both gastroscopy and colonoscopy failed to detect the primary tumor. An abdominopelvic CT scan was normal, but a CT scan of the chest disclosed a nodule measuring 2.5 x 1.5 cm in the lower lobe of the right lung. On bronchoscopy, it was found to be an invasive adenocarcinoma, consistent with a primary tumor of the lung. The patient was a heavy smoker (45 pack-years). The patient received 4 cycles of combined chemotherapy with carboplatine and gemcitabine, with no improvement. A month later, the patient complained of abdominal pain. Following demonstration of intra-abdominal spread of disease by CT scan, a second line chemotherapy was instituted with paclitaxel. A month later the patient's condition deteriorated and he complained of cough, sweating, and pain along the right leg. A bone scan revealed bone metastases in the right femur and left tibia. Two weeks later he was admitted to the hospital with intestinal obstruction and underwent laparotomy. He had massive intra-abdominal spread of cancer and ascites. Only a palliative colostomy was performed. The patient died 3 weeks later, 9 months after the diagnosis of adenocarcinoma of the lung. The clinical data on the three patients are summarized in Table I.

Macrophage-Induced Lymphangiogenesis and Metastasis following Paclitaxel Chemotherapy Is Regulated by VEGFR3.

While chemotherapy strongly restricts or reverses tumor growth, the response of host tissue to therapy can counteract its anti-tumor activity by promoting tumor re-growth and/or metastases, thus limiting therapeutic efficacy. Here, we show that vascular endothelial growth factor receptor 3 (VEGFR3)-expressing macrophages infiltrating chemotherapy-treated tumors play a significant role in metastasis. They do so in part by inducing lymphangiogenesis as a result of cathepsin release, leading to VEGF-C upregulation by heparanase. We found that macrophages from chemotherapy-treated mice are sufficient to trigger lymphatic vessel activity and structure in naive tumors in a VEGFR3-dependent manner. Blocking VEGF-C/VEGFR3 axis inhibits the activity of chemotherapy-educated macrophages, leading to reduced lymphangiogenesis in treated tumors. Overall, our results suggest that disrupting the VEGF-C/VEGFR3 axis not only directly inhibits lymphangiogenesis but also blocks the pro-metastatic activity of macrophages in chemotherapy-treated mice.

Differential therapeutic effects of anti-VEGF-A antibody in different tumor models: implications for choosing appropriate tumor models for drug testing.

We previously reported that the host response to certain chemotherapies can induce primary tumor regrowth, angiogenesis, and even metastases in mice, but the possible impact of anti-VEGF-A therapy in this context has not been fully explored. We, therefore, used combinations of anti-VEGF-A with chemotherapy on various tumor models in mice, including primary tumors, experimental lung metastases, and spontaneous lung metastases of 4T1-breast and CT26-colon murine cancer cell lines. Our results show that a combined treatment with anti-VEGF-A and folinic acid/5-fluorouracil/oxaliplatin (FOLFOX) but not with anti-VEGF-A and gemcitabine/cisplatinum (Gem/CDDP) enhances the treatment outcome partly due to reduced angiogenesis, in both primary tumors and experimental lung metastases models. However, neither treatment group exhibited an improved treatment outcome in the spontaneous lung metastases model, nor were changes in endothelial cell numbers found at metastatic sites. As chemotherapy has recently been shown to induce tumor cell invasion, we tested the invasion properties of tumor cells when exposed to plasma from FOLFOX-treated mice or patients with cancer. While plasma from FOLFOX-treated mice or patients induced invasion properties of tumor cells, the combination of anti-VEGF-A and FOLFOX abrogated these effects, despite the reduced plasma VEGF-A levels detected in FOLFOX-treated mice. These results suggest that the therapeutic impact of antiangiogenic drugs varies in different tumor models, and that anti-VEGF-A therapy can block the invasion properties of tumor cells in response to chemotherapy. These results may implicate an additional therapeutic role for anti-VEGF-A when combined with chemotherapy.

Low-dose metronomic chemotherapy: from past experience to new paradigms in the treatment of cancer.

Low-dose metronomic (LDM) chemotherapy represents an emerging concept in the treatment of cancer. Directed against tumor cells and other types of cells, such as endothelial and immune cells, this treatment regimen alters the tumor microenvironment and suppresses innate features which support tumor growth. Ongoing Phase III clinical studies explore various applications of LDM chemotherapy, mostly combined with other anticancer agents, to act as complementary treatments to conventional maximum tolerated dose (MTD) chemotherapy. In this article we summarize preclinical and clinical experience with LDM chemotherapy, emphasizing the potential contribution of this new treatment modality to future paradigms in the systemic treatment of patients with cancer.

Host response to short-term, single-agent chemotherapy induces matrix metalloproteinase-9 expression and accelerates metastasis in mice.

Mounting evidence suggests that bone marrow-derived cells (BMDC) contribute to tumor growth, angiogenesis, and metastasis. In acute reactions to cancer therapy, several types of BMDCs are rapidly mobilized to home tumors. Although this host reaction to therapy can promote tumor regrowth, its contribution to metastasis has not been explored. To focus only on the effects of chemotherapy on the host, we studied non-tumor-bearing mice. Plasma from animals treated with the chemotherapy paclitaxel induced angiogenesis, migration, and invasion of tumor cells along with host cell colonization. Lesser effects were seen with the chemotherapy gemcitabine. Conditioned medium from BMDCs and plasma from chemotherapy-treated mice each promoted metastatic properties in tumor cells by inducing matrix metalloproteinase-9 (MMP9) and epithelial-to-mesenchymal transition. In mice in which Lewis lung carcinoma cells were injected intravenously, treatment with paclitaxel, but not gemcitabine or vehicle, accelerated metastases in a manner that could be blocked by an MMP9 inhibitor. Moreover, chimeric mice reconstituted with BMDC where MMP9 activity was attenuated did not support accelerated metastasis by carcinoma cells that were pretreated with chemotherapy before their introduction to host animals. Taken together, our findings illustrate how some chemotherapies can exert prometastatic effects that may confound treatment outcomes.

Daily low-dose/continuous capecitabine combined with neo-adjuvant irradiation reduces VEGF and PDGF-BB levels in rectal carcinoma patients.

UNLABELLED: Metronomic low-dose chemotherapy regimen was found to have an antiangiogenic effect in tumors. However, its effect on levels of circulating pro-angiogenic and anti-angiogenic factors is not fully explored. MATERIALS AND METHODS: The levels of both VEGF and PDGF-BB were measured in three time points, in the serum of 32 rectal carcinoma patients receiving daily reduced-dose/continuous capecitabine in combination with preoperative pelvic irradiation. RESULTS: We found a significant decrease in VEGF and PDGF-BB serum levels during the combination treatment (p < 0.0001), followed by an increase in the successive rest-period (p < 0.0001). In addition, substantial changes in platelets counts were observed during treatment in correlation with the changes of VEGF and PDGF-BB serum levels. DISCUSSION: These results suggest that combined chemo-irradiation affect levels of pro-angiogenic factors during treatment, and may reflect an anti-angiogenic window induced during this treatment. The potential implications of this inducible phenomenon, including a possible clinical benefit from the administration of long lasting metronomic chemotherapy immediately following combined chemo-irradiation, would warrant further investigation.

Preoperative chemoradiation in rectal cancer: Retrospective comparison between capecitabine and continuous infusion of 5-fluorouracil.

BACKGROUND: We compared the efficacy and toxicity of oral capecitabine and continuous infusion of 5-fluorouracil (5-FU) in the preoperative chemoradiation treatment of patients with rectal cancer. PATIENTS AND METHODS: The files of 89 patients with rectal cancer, 43 treated preoperatively with oral capecitabine and 46 with intravenous 5-FU, were reviewed, and the outcome of the groups was compared. RESULTS: There was no statistically significant difference in the complete pathological response rate between the capecitabine and the 5-FU groups (30% vs. 17%, P = 0.15). The downstaging rate was higher in the capecitabine group (77% vs. 50%, P = 0.009). Toxicity was mild in both groups. The rate of Grade 3 gastrointestinal toxicity was similar in the two groups (diarrhea 2% vs. 4%, proctitis 5% vs. 7%), except for one patient in the 5-FU group (2%) who developed a rectovaginal fistula. In the capecitabine group, one patient (2%) had Grade 3 hand-foot syndrome, and another had an acute myocardial infarction. In the 5-FU group, two patients (4%) had Grade 3 hematological toxicity, and three (6%) had complications from Port-a-Cath insertion. CONCLUSION: Preoperative chemoradiation with oral capecitabine appears to be safe and well tolerated, and at least as good as continuous 5-FU.

Non-resectable slow-growing meningiomas treated by hydroxyurea.

PURPOSE: To test the benefit of hydroxyurea in the treatment of recurrent and non-resectable slow-growing meningiomas. METHODS: Twelve patients with regrowing non-malignant meningiomas, were enrolled for a protocol of 2 years with continuous chemotherapy with hydroxyurea, 20 mg/kg/day. Response to treatment was evaluated both clinically and by diagnostic imaging using computed tomography (CT) and 201-Thallium single photon emission CT. One minimal response was documented by CT, accompanied by clinical stabilization. Nine patients showed progressive disease, at least by one imaging procedure, with a median time to progression of 13 months (range 4-24). Two other patients were not available for response due to early removal from the study, following abrupt manifestation of grades 3-4 hematological toxicity. CONCLUSION: In this series hydroxyurea has not shown effectiveness in the treatment of non-resectable slow-growing meningiomas: neither for achieving response, nor for arresting disease progression.