Using DAB4, we examined La expression in human lung cancer samples to assess its suitability as a cancer-selective therapeutic target. We evaluated the safety and effectiveness of radioimmunotherapy (RIT) using DAB4 radiolabeled with Lutetium-177 , when delivered alone or after chemotherapy. PARPi  was combined with chemotherapy and the effects of combined treatment on tumour growth, tumour cell DNA damage and death, and intratumoural DAB4 binding were also analysed. The effect of the triple combination of PARPi, chemotherapy and 177Lu-DAB4 on tumour growth and survival of LL2 tumour-bearing mice was tested.The expression of La mRNA in human lung cancer samples was analysed using the online database Oncomine, and the protein expression of La was examined using a TissueFocus Cancer Survey Tissue Microarray. The binding of DAB4 to cisplatin-treated LL2 cells was assessed in vitro. An anti-tumour dose response was observed when escalating doses of 177Lu-DAB4 were delivered in vivo, with supra-additive responses observed when chemotherapy was combined with 177Lu-DAB4. Combining PARPi with chemotherapy was more effective than chemotherapy alone with increased tumour cell DNA damage and death, and intratumoural DAB4 binding. The combination of PARPi, chemotherapy and 177Lu-DAB4 was well-tolerated and maximised tumour growth delay.La was over-expressed at both mRNA and protein levels in surgical specimens of human lung cancer and the over-expression of La mRNA conferred a poorer prognosis. DAB4 bound specifically to cisplatin-induced dead LL2 cells in vivo, particularly after chemotherapy. Tumour uptake of DAB4 increased further after the combination of PARPi and chemotherapy, which generated new dead tumour cell-binding targets. Consequently, combining 177Lu-DAB4 with PARPi and chemotherapy produced the greatest anti-tumour response. Therefore, the triple combination of PARPi, chemotherapy and RIT may have broad clinical utility.The La antigen represents a dead cancer cell-specific target in lung cancer, and DAB4 specifically targeted tumour tissue  EGFR) gene and fusions of the anaplastic lymphoma kinase (ALK) gene, has yielded targets for small-molecule kinase inhibitors, which often produce regressions and durable tumour control[Lung cancer, particularly the common non-small cell lung cancer (NSCLC) variant, is one of the major contributors to cancer death worldwide. Moreove control. Neverth control. Further control.Targeting tumours with therapeutic monoclonal antibodies (mAbs) has proven to be an effective approach to cancer therapy although its clinical effectiveness has been more limited in NSCLC with the vascular endothelial growth factor-neutralising mAb, bevacizumab, the only approved therapy. Hence, lung cancer is relatively bereft of cell-surface targets for antibody therapies. The anti-tumour potency of mAbs can be improved by arming mAbs with radionuclides and cytotoxic drugs, creating the new therapeutic modalities of radioimmunotherapy (RIT) and antibody drug conjugates, respectively. Although armed mAbs usually target tumour cell-surface antigens, they can also elicit bystander killing of surrounding, antigen-negative tumour cells,6. Succein vivo unlike dead normal cells[in vivo[90Y), we have tested the concept of targeting dead tumour cells as a way to deliver RIT to surrounding viable tumour cells in murine syngraft and human xenograft models. Compared to DAB4-directed RIT alone, prior treatment with DNA-damaging cytotoxic chemotherapy resulted in supra-additive anti-tumour responses, with the quality of the responses ranging from significant tumour growth delay in carcinoma models[The lupus-associated (La) antigen, also known by the HUGO Gene Nomenclature Committee approved name of Sjögren Syndrome B (SSB), is an abundant, essential, and ubiquitously expressed ribonucleoprotein that is revealed preferentially in dead tumour cells because La is over-expressed in malignancy-10 and dal cells. The La-[in vivo, particu[in vivo. Using Da models to sustaa models.The safety and efficacy of RIT in carcinoma models may be improved by altering the radionuclide used for RIT and by combining RIT with radio-sensitising drugs or small molecule enzyme inhibitors. Since c177Lu-DAB4 with or without radio-sensitising chemotherapy for treating mice harbouring the well-characterised chemo- and radio-resistant Lewis Lung (LL2) tumour. We hypothesised that adding a PARPi to the treatment regimen would sensitise tumour cells to chemotherapy and generate more intratumoural dead cell targets for DAB4 to bind. The increased tumour accumulation of 177Lu-DAB4 together with continued PARPi treatment would further sensitise tumour cells to 177Lu-derived β-emissions, resulting in greater anti-tumour activity.In this study, we analysed expression of La mRNA in normal and malignant lung tissues and the expression of La protein in surgical specimens from lung cancer patients. We used p value significance threshold set at <10-4.The online cancer database Oncomine version 4.4.3  was used to examine the mRNA expression of La in normal versus cancerous lung tissue, with a LL2 cells were cultured in RPMI-1640  with 5% FCS . DAB4 is a subclone of the anti-La 3B9 hybridoma originated by Dr Michael Bachmann which waA TissueFocus Cancer Survey Tissue Microarray  was dewaxed and endogenous peroxidase activity was blocked with 0.5% hydrogen peroxide in methanol. After washing in PBS, heat-induced antigen retrieval using citrate buffer (pH 6) was performed. The slide was allowed to cool and washed twice with PBS followed by blocking with 3% normal human serum. The slide was incubated with 0.6 μg/mL DAB4 overnight at room temperature, followed by biotinylated horse anti-mouse IgG  and strepavidin conjugated horseradish peroxidase . To visualise antibody binding, the microarray was incubated with 3′-diaminobenzide and counterstained with Mayer’s Haematoxylin.5) were treated with varying concentrations of cisplatin for 48 h, collected, washed with FACS buffer  and incubated with 10 μg/mL DAB4 or Sal5 for 15 min. Cells were washed and incubated with 2 μg/mL goat anti-mouse Alexa-488  for 15 min, washed further, incubated with 1 μg/mL DAPI (Sigma-Aldrich) and analysed by flow cytometry .LL2 cells /2, where a is the shortest diameter and b is the longest diameter of the tumour. Treatment commenced when tumours reached 30 to 50 mm3. Mice were monitored daily using a clinical record sheet with points allocated for physical observations such as visible tumour, ruffled coat, hunched posture, reluctance to move, diarrhoea, squealing when handled and weight loss. Body weight and tumour volume were measured every 2 days in the first week of treatment and daily thereafter. Mice were humanely euthanized when a clinical score of 5 was reached, weight loss was >15% (cf. day 1), or tumour volume was >600 mm3.All animal experiments were approved by the SA Pathology Animal Ethics Committee, Adelaide, and conducted following institutional ethical guidelines. Six- to 8-week-old female C57Bl/6 mice were injected subcutaneously in the right flank with 10177Lu . Radioimmunoconjugates were administered intravenously on day 3. The specific activity of radioimmunoconjugates ranged from 95 to 130 MBq/mg with >97% incorporation of 177Lu as determined by instant thin layer chromatography. The PARPi inhibitor Rucaparib  was diluted in 5% D-glucose in PBS for intraperitoneal injection at 1 or 2 mg/kg and administered daily on days 1 to 5, 30 min before chemotherapy or RIT. For in vivo antibody binding analysis, DAB4 was biotinylated with EZ-Link NHS-Biotin (Thermo Fisher) following manufacturer’s instructions. One hundred micrograms of biotin-DAB4 was administered 24 h after chemotherapy.Tumour-bearing mice were treated intravenously with 50 mg/kg gemcitabine  on days 1 and 2 and 2.5 mg/kg cisplatin (Hospira) on day 1. DAB4 and Sal5 were conjugated to the bi-functional chelator DOTA-NHS  as previously described and radi2 gamma counter (PerkinElmer) with peak detection set at 208 keV. Radioactivity in the organs was normalized to the weight of the organ and the accumulation was calculated as the percentage of radioactivity per gram over the radioactivity of the injected dose of 177Lu-DOTA immunoconjugates (%ID/g). High-resolution digital autoradiography was performed on 4-μm tumour sections using a MicroImager .Mice were euthanized 24 h after RIT administration, tissues were collected and weighed and radioactivity was measured using a Wallac 2470 wizardMice were euthanized 24 h after administration of biotin-DAB4, tumours were removed and snap frozen in OCT cryoprotectant . Sections were fixed with 10% neutral-buffered formalin, washed with PBS, blocked with 5% BSA with 0.3% Triton-X 100 (Sigma-Aldrich), washed and incubated overnight with rabbit anti-phospho-histone H2AX (Ser139) antibody . Sections were washed and incubated with donkey anti-rabbit IgG NL557 . To detect biotin-DAB4, sections were concurrently incubated with strepavidin-Alexa Fluor-488 . Cell death was detected using the ApopTag cell death detection kit  following manufacturer’s instructions. Sections were stained with 1 μg/mL DAPI, and examined using an Olympus IX71 microscope with CellSens Standard (v1.6) software and analysed using ImageJ (v1.45) software .Measurements of association between La mRNA expression and lung cancer outcomes were perp = 0.008). This ratio was very similar when adjusting for nodal status in a Mantel-Haenszel analysis at 1.27 , and heterogeneity testing showed similarly elevated rate ratios for both the node-positive and node-negative cases (p = 0.806).We used the Oncomine online gene expression database to examine the expression of La mRNA in human lung cancer samples compared to normal lung tissue. Two studies,30 demonThe expression of La protein in lung cancer samples was also investigated by immunohistochemistry using a cancer tissue microarray stained with the La-specific mouse mAb, DAB4 and scored by a pathologist. Only nuclear staining with DAB4 was considered positive and scoring was based on staining intensity  and percentage of positively stained cells. In the 10 lung cancer sections, all demonstrated strong staining; in two sections, >95% tumour cells were positive, and in the remaining eight sections, >99% tumour cells were positive. In the 5 normal lung tissue sections, approximately 50% cell nuclei had weak to moderate DAB4 staining, mainly belonging to pneumocytes and endothelial, inflammatory and stromal cells. As illustrated in Figure in vitro with escalating doses of cisplatin for 48 h and binding of DAB4 or its isotype control mAb  to dead (DAPI+) cells was examined. Cisplatin treatment resulted in a dose-dependent increase in cell death  from 8 days for untreated mice to 10, 12 and 12 days after 5, 7.5 and 10 MBq 177Lu-DAB4, respectively . The combination of chemotherapy and 10 MBq 177Lu-DAB4 was not tolerated, and the entire cohort was euthanized 16 days after treatment because 60% of the treatment group had a clinical score of 5. None of mice in the remaining treatment groups had evident toxicity, with only minimal and transient body weight loss observed  compared to mice which received only chemotherapy and 5 MBq 177Lu-DAB4 (MST of 21 days). Analysis of the TDT for each treatment group demonstrated that the addition of PARPi to the treatment regimens of RIT, chemotherapy or combination of chemotherapy and RIT resulted in an increase in TDT greater than would be expected if the addition of PARPi was only additive  suggest that a saturating dose for 177Lu-DAB4 monotherapy had been reached, perhaps because of a limiting number of DAB4-binding dead tumour cell targets[177Lu-DAB4 resulted in a supra-additive anti-tumour response, and reflected the similar supra-additive response observed with combined chemotherapy and 90Y-DAB4, which we characterised as a ‘genotoxic chain reaction’[90Y-DAB4 and 177Lu-DAB4 behaved as residualizing radioimmunoconjugates after combination chemotherapy and RIT (chemo-RIT): intratumoural, detergent-resistant 90Y-DAB4 was found 96 h post-chemo-RIT[177Lu-DAB4 was found in LL2 tumours 24 h post-chemo-RIT. The complex mechanism involved in the supra-additive responses may depend on at least two factors:As a monotherapy,  targets. The comeaction’. Moreovehemo-RIT, and 177177Lu-DAB4 results in radiation crossfire as 177Lu-generated β-particles can kill cells up to 50 cell diameters from antibody-bound target cells[(i) increased chemotherapy-induced tumour cell death with the associated increase in intratumoural binding of et cells, thus ge177Lu-DAB4 to bind thereby permitting continued irradiation of surrounding viable tumour cells.(ii) the newly generated dead tumour cells provide additional targets for circulating 177Lu-DAB4 alone or with chemotherapy was higher than that observed for 90Y-DAB4 (MTD of 5 MBq) in the LL2 tumour model[177Lu and 90Y are used for clinical RIT, we suggest that 177Lu may have combined safety and efficacy advantages over 90Y as a therapeutic β-emitter, at least in treating small tumour volumes[177Lu vs90Y have a lower maximum energy (0.5 MeV with 79% occurrence vs 2.2 MeV with 99% occurrence), lower dose-rate constant (0.076 and 0.54 Gy.g/MBq.h), shorter range (maximum tissue penetration 2 mm vs 11 mm), but a longer half-life (6.71 days vs 2.67 days). Therefore, the likelihood that the radiation dose absorbed by susceptible tissues such as gut and bone marrow will be lower and that more radiation dose will be absorbed for longer within the confines of the shrinking tumour favours 177Lu-DAB4 rather than 90Y-DAB4 for La-targeted RIT post-chemotherapy. Consequently, we introduce the concept of ‘volume-adapted radioimmunotherapy’ to describe the match between the physical characteristics of 177Lu and tumour size post-chemotherapy treatment.Furthermore, the MTD of ur model. As both volumes. β-partiAs the combination of PARPi with chemotherapy or radiotherapy has been shown to have greater potency than each individual treatment, we reasp53[p53 expression reduces sensitivity to PARPi[177Lu-labeled anti-human EGFR mAb). Here, complete eradication of pulmonary metastases and patient-derived xenografts of human triple-negative breast cancer was achieved in immunodeficient mice. This effect of triple therapy was attributed to an increase in tumour cell death and killing of putative breast cancer stem cells[Nevertheless, tumours were not eradicated after a single cycle of triple combination therapy. Among the possible explanations is that the LL2 cell line is known to have mutant p53, and altto PARPi. Hence, to PARPi, could bem cells, which m177Lu as the payload, we have shown that La-targeted RIT is well tolerated and able to inhibit growth of LL2 tumour syngrafts pre-treated with chemotherapy. Moreover, the addition of a PARPi potentiated the anti-tumour effects of both chemotherapy and RIT. Additional approaches such as repeated cycles of RIT, conjugation to α-emitting radionuclides, or chemo-RIT combinations with inhibitors of EGFR signalling, cell cycle checkpoints, or DNA repair, may contribute to eradication of LL2 tumours. Nonetheless, here we demonstrate the principle that bystander killing by a short-range β-emitter is a feasible and active approach to the treatment of lung cancer. This approach may find clinical application as consolidation treatment for such standard treatments as platinum-based chemotherapy and radiotherapy in advanced disease and adjuvant settings.In conclusion, using RIT with MP Brown, F Al-Ejeh and JM Darby are co-inventors on APOMAB-related patents both issued and pending.in vivo experiments and data collection. DMR performed data analysis comparing survival of NSCLC patients with La expression and performed statistical analysis. AR analysed the Tissue microarray and JM developed and performed immunohistochemistry and imaging of the Tissue microarray. MPB participated in the design of the study, data collection, analysis and interpretation, and in writing the manuscript. All authors read and approved the final manuscript.AHS designed and performed experiments, collected the data, performed statistical analysis and wrote the paper. FA designed and performed experiments, collected the data and performed statistical analysis. CKF and JMD were involved with (A) or chemotherapy and RIT (B) are shown, n = 5.Mouse body weight change after treatment with RIT alone or in combination with chemotherapy. Figure S1 Mice were treated with RIT alone or with chemotherapy (chemo) as described in Methods. The percent change in mouse weights for mice treated with RIT alone Click here for file177Lu-DAB4 or 90Y-DAB4 alone or in combination with chemotherapy. Figure S2 Mean tumour doubling times (TDT) for tumour-bearing mice treated with (A) 177Lu-DAB4 or (B) 90Y-DAB4 alone or combined with chemotherapy were derived from tumour growth curves. The broken line represents the TDT if the combination of chemotherapy and RIT were additive. Data from (B) has been published previously in[LL2 tumour doubling time after treatment with ously in.Click here for file(A) or PARPi and chemotherapy (B) are shown, n = 4–5.Mouse body weight changes after treatment. Figure S3 Mice were treated with PARPi inhibitor (1 or 2 mg/kg) alone or with chemotherapy (chemo) as described in Methods. The percent change in mouse weights for mice treated with PARPi alone Click here for filePARPi + RIT = TDTRIT + (TDTPARPi – TDTuntreated).LL2 tumour doubling time after treatment regimens including PARPi, chemotherapy or RIT. Figure S4 Mean tumour doubling times (TDT) for tumour-bearing mice in each treatment group were derived from tumour growth curves. Filled bars indicate increases in TDT that would be expected from adding the effects of component treatments, whereas open bars indicate the observed TDT for treatments. Additive effects were calculated by deducting the TDT of the untreated group from the TDT of the PARPi treatment group and adding that to the TDT of the group of interest, e.g. TDTClick here for file


There is a typographical error in the funding statement. Grant NS060885 is incorrectly published as NS06885.


In the E-mail list, guoyahong7225@163.com (YG) should be in front of maozuguo@hit.edu.cn (MG). The E-mail list should read:"*E-mail: guoyahong7225@163.com (YG); maozuguo@hit.edu.cn (MG); yufei.huang@utsa.edu (YH)"Posted by the authors of this article.


The author analyzes a historical, long, and tortuous way to discover the cerebrospinal fluid. At least 35 physicians and anatomists described in the text have laid the fundamentals of recognition of this biological fluid's presence. On the basis of crucial anatomical, experimental, and clinical works there are four greatest physicians who should be considered as equal cerebrospinal fluid's discoverers: Egyptian Imhotep, Venetian Nicolo Massa, Italian Domenico Felice Cotugno, and French François Magendie. Cerebroπνεΰμα  ψυχικóv, Latin: spiritus animalis) with its mental functions was located within the cerebral ventricles  ad motum liquidi per Nervos” and then out of the brain to the spinal cord “autem cerebrum & spinalem medullam” . Work byIn a description of the brain anatomy with precise pictures of brain structures published in 1684 Raymundi Vieussens, French anatomist, referred to Galen's theory on 35 pages. He gave careful consideration to “spiritus animalis” in separate chapters , where he attributed all body movements to this phenomenon . AccordiTractatus)  a sanguine derivatum fluidum” , 44. AgaAqua ventriculorum, from the Latin edition of Haller's 1762 Elementa Physiologiae Corporis Humani  Egypt […] Moreover, everyone in the whole country is a skilled physician” [It has been pointed out that even Homer in his epic poem ysician” . Shehataysician” . And whaysician” .in vivo in 3000 B.C. The description of the discovery was found in The Papyrus of Smith of 1600 B.C.The Egyptian physician Imhotep is the most likely to be the first one to discover intracranial cerebrospinal fluid Liber Introductorius Anatomiae was the first one to describe cerebrospinal fluid within cerebral ventricles based on the postmortem autopsies.Nicolo Massa in his 1536 publication De Ischiade Nervosa Commentarius of 1764 was the first one to describe cerebrospinal fluid in the subarachnoid space around spinal cord.There is historical and consensual agreement that Domenico Cotugno was the first one to discover cerebrospinal fluid through the experimental postmortem research during the autopsies. Cotugno in his The name “cerebrospinal fluid” was introduced by François Magendie in the first half of the 19th century. Magendie was the first to discover method of cerebrospinal fluid pressure measurement and he was able to lay the scientific foundation for development of the cerebrospinal fluid dynamic research.


Human carcinomas frequently exhibit significant stromal reactions such as the so-called “desmoplastic stroma” or “reactive stroma”, which is characterised by the existence of large numbers of stromal cells and extracellular matrix proteins. Carcinoma-associated fibroblasts (CAFs), which are rich in activated fibroblast populations exemplified by myofibroblasts, are among the predominant cell types present within the tumour-associated stroma. Increased numbers of stromal myofibroblasts are often associated with high-grade malignancies with poor prognoses in humans. CAF myofibroblasts possess abilities to promote primary tumour development, growth and progression by stimulating the processes of neoangiogenesis as well as tumour cell proliferation, survival, migration and invasion. Moreover, it has been demonstrated that CAFs serve as a niche supporting the metastatic colonisation of disseminated carcinoma cells in distant organs. Their contribution to primary and secondary malignancies makes these fibroblasts a potential therapeutic target and they also appear to be relevant to the development of drug resistance and tumour recurrence. This review summarises our current knowledge of tumour-promoting CAFs and discusses the therapeutic feasibility of targeting these cells as well as disrupting heterotypic interactions with other cell types in tumours that may improve the efficacy of current anti-tumour therapies. Tumours are a complex tissue composed of carcinoma cells, various types of stromal cells and dense extracellular matrix (ECM). Recent studies have indicated the tumour-promoting roles of stromal cells, as exemplified by vascular cells, immune cells, fibroblasts, myofibroblasts, adipocytes and bone marrow-derived progenitors ,3,4,5,6.Considerable numbers of CAFs are frequently observed within the tumour-associated stroma of various human cancers, including those of the breast, prostate, lung, colon and pancreas ,15. ThesIn addition to the ability of CAFs to promote neoangiogenesis and tumour growth ,26,27,28Both activated and non-activated fibroblasts comprise CAF populations in the tumour-associated stroma. Activated stromal fibroblast populations in these cells have often been identified by the expression of several markers including α-SMA ,33, tena+) bone marrow prior to subcutaneous implantation of murine ovarian carcinoma cells. Importantly, the majority of FSP-1+ and FAP+ fibroblasts in the resulting tumour xenografts were shown to derive largely from GFP+ bone marrow cells including MSCs. In contrast, α-SMA+ and NG2+ myofibroblasts in tumour started to develop mainly from RFP+ local resident tissue exemplified by the adipose tissue [CAFs are also known to originate from heterogeneous cell types including resident fibroblasts, preadipocytes, endothelial cells and bone marrow-derived cells, such as mesenchymal stem cells (MSCs) and fibrocytes ,41,42. Ae tissue .Taken together, these findings suggest that CAFs are composed of various fibroblast populations with their distinct cellular origins. Continuous infiltration of a tumour mass by these various fibroblast progenitors, and their subsequent transdifferentiation into CAFs, can facilitate the generation and expansion of the so-called desmoplastic, reactive stroma that in turn supports tumour progression. Potential diverse tumour-promoting functions of the distinct fibroblast populations within CAFs remain to be addressed in future studies.de novo production and incorporation of α-SMA into stress fibres, exhibit a greater ability to contract collagen gels in vitro. Our previous work demonstrated that the greater ability of CAFs to contract collagen gels in culture correlated significantly with their ability to more effectively promote the growth of co-implanted human breast carcinoma cells in recipient mice [Activated myofibroblasts, which are characterised by the ent mice . This fiin vitro propagation without the ongoing interaction with carcinoma cells [in vivo study employing experimentally generated models of kidney fibrosis demonstrated that DNA methylation involves maintaining the stability of fibroblast activation [Notably, the activated myofibroblastic trait and the tumour-promoting phenotypes of CAFs were stably maintained during their ma cells ,38. Primma cells ,44,45. Itivation . Transfotivation . The resIt has been shown and widely accepted that carcinoma cells and stromal cells co-evolve during the course of tumour progression ,47; carcIn addition to others, our study showed that subcutaneous co-implantation of human mammary stromal fibroblasts with breast carcinoma cells into recipient mice resulted in transdifferentiation of these fibroblasts into tumour-promoting CAF myofibroblasts during tumour progression . These eα-SMA-negative (or negligible) PDGFR-α-positive stromal cells extracted from the neoplastic skin of K14-HPV16 (HPV) transgenic mice were also characterised as CAFs . Their tloxP/loxP transgenic mice [+) cells that form a subpopulation of stromal fibroblasts within CAFs. Loss of Pten expression in FSP-1+ CAFs resulted in an accelerated growth rate of ErbB2-positive mammary carcinoma. This was suggested to be a consequence of expansion of the desmoplastic stromal reaction involving numerous infiltrating macrophages. Notably, the Pten-deficient stromal fibroblasts upregulated Ets 2 expression and downregulated miR-320, a negative regulator of Ets 2 expression that contributed to increased mammary tumour growth [+ stromal fibroblasts serves as a negative regulator of Ets2 expression via miR-320 to inherently constrain mammary tumourigenesis.Pten is one of the main regulators of PI3K signalling and is a tumour suppressor with lipid and protein phosphatase activity ,52. The nic mice . In thisr growth . Thus, ir growth ,53. Collin vivo [Recent studies have also shown that carcinoma cell-derived hedgehog (Hh) ligands induce the Smo signalling pathways in the surrounding stroma in a paracrine fashion ,54,55. Hin vivo ,54,55. TIt is assumed that CAFs sustain their various aforementioned tumour-promoting signalling pathways without an ongoing interaction with carcinoma cells. The persistence of CAF phenotypes is presumably achieved through the acquisition of genetic and/or epigenetic alterations in these cells. Recent studies using genome-wide comparative genomic hybridisation and single-nucleotide polymorphism array analyses indicated that clonal somatic genetic alterations are very rarely detected in tumour stroma present within human cancers ,58,59,60Furthermore, understanding of the consequences of altered profiles of DNA methylation within CAF genomes ,65, idenIn addition to the primary tumour-promoting function of CAFs, recent studies have demonstrated an ability of these cells to serve as a niche for CSCs relevant to the promotion of tumour growth, invasion and metastasis ,66,67,68CCL2  is also a well-known potent chemoattractant for monocytes and other immune cells which migrate to areas of inflammation and tumour. This chemokine, produced by CAFs, was reported to confer invasive and CSC phenotypes on carcinoma cells by acting directly through CCR2 and CCR4 receptors ,71,72. CAnother study also indicated that CAFs are responsible for inducing epithelial to mesenchymal transition (EMT) and the associated CSC phenotypes in human prostate carcinoma cells . Two difTumour-associated stromal cells interacting with ECM proteins also modulate intracellular adhesions and cell contractility, thereby contributing to the generation of tensile forces in the tumour microenvironment that can promote breast cancer progression ,74. It h−/− background as compared to the control POSTN+/+ background [+CD90+ CSC-enriched cancer cell proportions and mammosphere formation, and the addition of POSTN reversed the decreased mammosphere formation in MMTV-PyMT POSTN−/− tumour cells. Furthermore, stromal POSTN was shown to interact with Wnt ligands to augment the presumed signalling activity. Taken together, these findings demonstrated that CAF-produced POSTN in the lung drives metastatic colonisation of disseminated breast carcinoma cells via induction of Wnt signalling and thus the CSC trait.The colonisation of distant organs by disseminated carcinoma cells is one of the most crucial steps in the multi-step process of tumour metastasis. This metastatic colonisation by cancer cells largely depends on the support provided by the stromal niche, which is created by a unique composition of local stromal environmental factors including ECM periostin (POSTN) and tenascin-C (TN-C) proteins ,77,78. Mckground . Moreove+ stromal fibroblasts that contribute to producing TN-C were eradicated in transgenic mice upon expression of viral thymidine kinase under the control of the FSP-1-promoter and with ganciclovir treatment [TN-C, as a crucial component of the metastatic environment, is also an appealing target for new therapeutic strategies. FSP-1reatment . As a rereatment . SimilarTargeting the tumour-associated stroma is believed to be essential for the development of new and effective cancer therapies ,12,13,79It has been reported that the ability of these fibroblasts to remodel the ECM and generate tracks for carcinoma cell migration is associated with their collagen gel contractile ability . A recenin vivo studies using tumour xenografts and transgenic tumour mouse models also showed that inhibition of PDGFR signalling in CAFs facilitates intratumoural drug delivery [The activation of PDGFR signalling is known to be crucial for CAFs to conduct their tumour-promoting function ,102. PDGdelivery ,107. Thedelivery ,107.In addition to PDGF-PDGFR signalling, Hh-Smo signalling also plays important roles in modulating intratumoural drug delivery. Hh ligands, which are released from carcinoma epithelial cells, induce Smo activity via binding to the PTCH1 receptor expressed on the surrounding stroma, as mentioned earlier . EffectsN-acetylglucosamine and glucuronic acid [An ECM component known as hyaluronan  abundant in tumour stroma is a simple polysaccharide molecule composed of repeating disaccharide units of alternating nic acid . The dynnic acid . A recennic acid . It has +myeloid-derived suppressor cells, CD4+Foxp3+ T regulatory cells and MSCs. Several recent studies have also indicated that CAFs contribute to tumoural immune suppression [Immune suppressive roles of the tumour microenvironment that prevent suppression of tumour growth have widely been accepted ,110. Thepression ,81,95.+ T cells into the tumour. Moreover, treatment with the DNA vaccine together with doxorubicin substantially increased intratumoural uptake of the cytotoxic drug and prolonged lifespans of the vaccinated mice [FAP is a type II transmembrane protein that functions as a post-prolyl protease. The expression of this protein is also detected on the cell surfaces of activated fibroblast populations of CAFs. FAP-null mice in fact showed a decrease in the tumourigenicity of endogenous K-rasG12D-driven lung carcinoma cells and syngeneic colon tumour cells . Targetited mice . Collect+) CAFs were further studied using a transgenic mouse expressing the diphtheria toxin receptor gene under the control of the 217 kb FAP gene promoter region cloned into a bacterial artificial chromosome [+ CAFs were selectively eliminated by administering diphtheria toxin to transgenic mice bearing Lewis lung carcinoma cells expressing ovalbumin that rendered the cells immunogenic. As a result, the subcutaneous immunogenic tumour showed substantially attenuated growth. The decreased tumour growth was also mediated by induction of rapid hypoxic necrosis of both cancer and stromal cells without altering the T cell proportions present in the tumour. Moreover, neutralising antibodies against tumour necrosis factor-α or interferon-r restored tumour growth inhibition by ablating FAP+ CAFs, indicating essential roles of these fibroblasts in modulating carcinoma cell responses to the cytotoxic effects of both cytokines. Taken together, these data support the notion that FAP+ CAFs play immunosuppressive roles in promoting the tumourigenesis of immunogenic tumour cells.The biological roles of FAP-positive  engrafted into immunocompromised mice have been documented to reflect tumour histopathology, growth and disease outcomes [in vivo imaging technology and extracting them for subsequent in vitro analyses will allow us to evaluate the efficacy and specificity of CAF-targeting anti-tumour therapies. Further implantation of human immune cells may be also considered in efforts to render the PDTX mouse partially immunocompetent [It has become apparent that CAFs serve as a niche promoting tumour growth and progression by conferring invasive, metastatic and CSC phenotypes upon neighbouring carcinoma cells. There have been reports on a number of tumour-promoting signalling pathways that mediate the interactions between CAFs and different cell types existing within tumours. Targeting the CAF-orchestrated tumour-prone microenvironment should therefore be applicable to developing novel cancer treatments. This, however, remains a challenging task due to the presence of distinct CAF populations expressing α-SMA, TN-C, FAP, FSP-1  and/or PDGFR-α/β. Targeting a particular CAF population in genetically engineered mouse tumour models may not fully reveal the biological roles of all CAF populations during tumourigenesis. Moreover, these molecular markers can’t distinguish activated fibroblasts within tumour from these cells also present in non-cancerous tissues. This would thus make the specific targeting of the tumour-associated fibroblasts outcomes ,114. It ompetent .Several multi-targeted anti-angiogenic tyrosine kinase inhibitors  against vascular endothelial growth factor receptors (VEGFRs), PDGFRs, fibroblast growth factor receptors (FGFRs) and/or epidermal growth factor receptors (EGFRs), showed a greater anti-tumour efficacy than single-target drugs in patients, likely reflecting the facilitated acquisition of drug resistance . Recent


According to the practice guidelines of the American Burn Association on burn shock resuscitation, intravenous (IV) fluid therapy is the standard of care for the replacement of fluid and electrolyte losses in burn injury of ≥20% of the total body surface area. However, in mass burn casualties, IV fluid resuscitation may be delayed or unavailable. Oral rehydration therapy (ORT), which has been shown to be highly effective in the treatment of dehydration in epidemics of cholera, could be an alternate way to replace fluid losses in burns. A prospective case series of three patients was carried out as an initial step to establish whether oral Ceralyte®90 could replace fluid losses requiring IV fluid therapy in thermal injury. The requirement of the continuing IV fluid therapy was reduced by an average of 58% in the first 24 hours after the injury (range 37-78%). ORT may be a feasible alternative to IV fluid therapy in the resuscitation of burns. It could also potentially save many lives in mass casualty situations or in resource-poor settings where IV fluid therapy is not immediately available. Further studies are needed to assess the efficacy of this treatment and to determine whether the present formulations of ORT for cholera need modification. Oral rehydration therapy (ORT) was formulated for treating cholera in the 1960s . It was A prospective study of three patients with burn wounds (20-40% of TBSA) was performed. Fluid requirements were calculated according to the Parkland Formula  . For theAll the patients tolerated the administration of ORT with gastric residuals of less than 300 cc which were monitored every two hours. The characteristics of the patients are defined in We reviewed the literature on oral fluid replacement in burns but found only one report on the use of ORT in burns in humans, which treated burns 10-20%) in children. Resuscitation of IV fluid is the current standard of care for the treatment of patients with burns of ≥20% of the TBSA. Without intervention, the loss of fluids and electrolytes into burned tissues rapidly leads to hypotension and shock. In major burns, early resuscitation fluid is required to maintain circulation and adequate tissue perfusion. Volume losses occur most rapidly in the first 6-8 hours but continue for 18-36 hours or longer ,10. Howe-20% in cORT is a proven safe and effective alternative to IV resuscitation in epidemic cholera, a disease which causes rapid loss of fluids from the gut, leading to circulatory collapse and death. Oral rehydration solutions (ORS) take advantage of a robust back-up mechanism for intestinal absorption of salts and water, in addition to the normal absorption which is mediated by a sodium chloride-linked transport system at the brush border of the intestinal epithelium. This active transport moves sodium with chloride from the intestinal lumen into the cell. Cellular sodium potassium ATPase extrudes sodium into the lateral cell spaces, creating an osmotic and electrochemical gradient that produces a net flow of water and solutes from the lumen into the bloodstream ,12.This sodium-glucose transporter of the intestinal epithelium allows absorption of sufficient water and electrolytes to restore the large fluid losses even in severe diarrhoeal diseases when the normal absorptive mechanism is impaired . The WorIn the present limited case series, we have shown that ORT decreased the amount of IV fluids needed to maintain adequate hydration in patients with severe burn wounds. By introducing a rice-based ORT—Ceralyte®90, there is the potential to substantially decrease the IV fluid requirement in the resuscitation of the thermally-injured patient, thereby decreasing the risk of such complications as line infection. Furthermore, the absorption offluid and electrolyte from the intestine is autoregulated by homeostatic mechanisms, including endocrine and mechano receptors. ORT is, therefore, less likely to result in fluid overload which can cause significant complications, such as compartment syndrome and pulmonary oedema, occasionally observed in burned patients receiving IV fluid therapy .JAMA stated that “The use of oral saline is adopted as standard procedure in the treatment of shock due to burns, in the event of large scale civilian catastrophe”  used in this study.


Recently, small molecules termed ‘BH3-mimetics’ have been developed that mimic the action of pro-apoptotic BH3-only proteins. These bind and neutralize pro-survival proteins including BCL-2.Impairment of apoptosis is a hallmark of cancer and can result in resistance to chemotherapy. Tumour resistance to apoptosis is frequently acquired through deregulated expression of BCL-2 family members or inactivation of the p53 tumour suppressor pathway. Over-expression of the pro-survival protein BCL-2 is common in breast cancer (where it is readily detected by immunostaining), and has been shown to be an important prognostic marker. A potential role for BCL-2 as a Tissue microarrays containing 197 primary breast tumours were evaluated for the expression of BCL-2, its anti-apoptotic relatives MCL-1 and BCL-XL, and the pro-apoptotic BH3-only ligand BIM. These proteins were co-expressed at relatively high levels in a substantial proportion of heterogeneous breast tumours, including clinically aggressive basal-like cancers. To determine whether the BH3-mimetic ABT-737 that neutralizes BCL-2, BCL-XL and BCL-W, had potential efficacy in targeting BCL-2-expressing basal-like triple negative tumours, we generated a panel of primary breast tumour xenografts in immunocompromised mice and treated recipients with either ABT-737, docetaxel or a combination. Tumour response and overall survival were significantly improved by combination therapy, but only for tumour xenografts that expressed elevated levels of BCL-2. Treatment with ABT-737 alone was ineffective, suggesting that ABT-737 sensitizes the tumour cells to docetaxel. Combination therapy was accompanied by a marked increase in apoptosis and dissociation of BIM from BCL-2. Notably, BH3-mimetics also appeared effective in BCL-2-expressing xenograft lines that harbored p53 mutations.in vivo evidence that BH3-mimetics can be used to sensitize primary BCL-2-expressing breast tumours to taxane chemotherapy. Our results suggest that elevated BCL-2 expression constitutes a predictive response marker in breast cancer. These findings provide a rationale for the development of clinical protocols using the oral analogue ABT-263 (navitoclax) as an adjunct to taxane chemotherapy in BCL-2-expressing basal-like and luminal breast cancer.In summary, primary breast tumour xenograft models that recapitulate the phenotype of the primary tumour have been developed as useful ‘proof-of-principle’, pre-clinical models. Our findings provide the first


Varicella zoster virus (VZV) is an exclusively human highly neurotropic alphaherpesvirus. To date, VZV has been shown to induce apoptosis, primarily through the intrinsic pathway in different cell types, except for neurons in which the virus becomes latent. This review summarizes current studies of varicella-induced apoptosis in non‑neuronal cells. Future studies are proposed to determine whether apoptosis is terminated prematurely or even begins in neurons that are non-productively infected with VZV.  During apoptosis, caspases (cysteine proteases) are activated that cleave DNA and lead to cell death [in vivo and in vitro [Programmed cell death, ll death . Common in vitro . VZV is in vitro ,4,5. WitZoster is characterized by dermatomal distribution pain that lasts for many weeks. Unfortunately, pain often persists for months or years  after zoster. Other serious neurological complications of zoster include meningoencephalitis, myelitis, vasculopathy, zoster paresis and retinitis ,7. PreveAlthough there is no satisfactory model for VZV pathogenesis or latency in rodents or primates, VZV has a counterpart in the alphaherpesvirus SVV. Like VZV, SVV infection of non-human primates causes varicella, after which virus becomes latent in ganglionic neurons and reactivates to produce zoster ,9. VZV aUnlike necrosis (death due to extracellular trauma), apoptosis of infected cells limits virus spread and thus may be beneficial to the host . ProgramThe intrinsic pathway of apoptosis is triggered by intracellular signals. This pathway is mediated by members of the Bcl-2 superfamily that includes pro- and anti-apoptotic proteins. Anti-apoptotic proteins are Bcl-2, Bck-xL and Bcl-w; pro-apoptotic proteins include Bid, Bax, BAD, Bak and Bok . Pro-apoOther cell signaling pathways can also affect apoptosis. Because of their potential role in VZV pathogenesis, the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the family of mitogen-activated protein kinases (MAPK), represent a pathway of particular interest. This pathway is initiated by an extracellular growth factor that binds to its receptor. This binding triggers a cell signaling cascade in which cytosolic Ras protein converts GDP into GTP and phosphorylates Raf. Raf then phosphorylates MAP kinase MEK1/2. Finally, MEK1/2 phosphorylates ERK1/2, which then acquires various effector functions, including activation of transcription factors that mediate cell proliferation  and enhaVZV induces apoptosis in African monkey kidney Vero cells, human melanoma MeWo cells, human fibroblasts and peripheral blood mononuclear cells in culture ,17,18,19Although VZV- and SVV-infected cells become apoptotic, thus limiting spread of virus, studies of VZV proteins revealed that they can increase cellular proliferation and viral replication. For example, the protein encoded by VZV open reading frame (ORF) 12 activates AP1, a transcription factor that increases cellular proliferation . A role Since VZV becomes latent in neurons, the question arises whether apoptosis is terminated prematurely so that VZV does not kill neurons, or whether the apoptotic cascade even begins. Although VZV transcripts and proteins have been detected in latently infected human ganglia, there are no morphological changes in neurons to suggest apoptosis . While nVZV ORF63 is the most prevalent and abundant transcript found in latently infected human ganglia . IncreasFinally, while miRNAs can regulate pro- and anti-apoptotic gene expression levels through transcriptional degradation or translational control, VZV-specific miRNAs have not been detected in human ganglia latently with VZV . in vitro. Finally, our laboratory has generated an SVV ORF63 mutant that can be used to study the role of varicella IE63 in vivo.The fact that VZV reactivation produces multiple serious neurological disorders underscores the need for a more complete understanding of the VZV-host relationship in neurons and non-neuronal cells. We are currently searching for markers of early and late stages of the apoptotic cascade, as well as for markers indicative of autophagy in VZV-infected neurons in cultures that do not exhibit a cytopathic effect after experimental infection. We are also using VZV ORF63 mutant viruses to determine if VZV IE63 is anti-apoptotic


Infusing large volumes of 0.9% sodium chloride  causes hyperchloremic acidosis. The clinical relevance of this effect remains contentious and saline is still the most commonly used resuscitation fluid in the US. However, a recent trial showed that saline or albumin in saline increased mortality in children with malarial sepsis, compared to no fluid . Infusion = 25) or plasmalyte (n = 25) over 4 hours. Arterial blood gases, serum creatinine, urea, and lactate were measured at baseline, 18 hours after CLP (before resuscitation), after resuscitation, and 24 hours after resuscitation. Blood pressure and pulse rate were measured during fluid infusion.Fifty adult male Sprague-Dawley rats were subjected to CLP . Eighteen hours later, they were randomly assigned to receive either 30 ml/kg saline (P < 0.0001) and lower pH  compared to plasmalyte. In addition, lactate was significantly higher after fluid infusion in the saline group  compared to plasmalyte, despite being similar before infusion . However, neither mean arterial blood pressure  nor heart rate  differed between the two groups.Saline-treated animals developed significantly higher levels of serum chloride (111 mmol/l vs. 102 mmol/l, +, which is stabilized in the presence of chloride [Saline infusion worsens lactic acidosis, despite similar blood pressure, when compared to plasmalyte. The mechanisms responsible for this effect are unclear. However, deoxygenated hemoglobin readily binds hydrogen ions, forming HbHchloride . Consequ


Chylothorax is a rare clinical entity characterized by a milky white aspirate with increased triglyceride levels. The commonest etiology is malignancy and trauma, and bilateral chylothorax, secondary to tuberculosis, is an extremely rare cause, as observed in the present case. Chylothorax is a relatively rare cause of a pleural effusion and it occurs when chyle, characterized by high-triglyceride and low-cholesterol concentrations, is found in the pleural space and is usually associated with neoplasms or trauma to the thoracic duct.2 The devA 15-year-old female was admitted to our department with the complaints of breathlessness, and off and on fever for 1 month. She came from a nonendemic zone of filariasis in Uttar Pradesh. The resting pulse rate was 92/min and blood pressure was 112/74 mmHg. There was no history of contact with a tuberculosis patient. Her physical examination revealed hard, mobile lymph nodes of size 2 × 2 cm approximately, present bilaterally in the cervical region, and pallor. On chest examination, there was stony dull note localized to the bilateral infrascapular, lower axilla. Rest of the systems were within normal limits.3; differential leukocyte count was neutrophils 20, lymphocytes 80, pleural fluid triglyceride 535 mg%, and pleural fluid cholesterol 24.8 mg%; pleural fluid of the left side revealed protein 4.0 g%, sugar 40 mg%, and total leukocyte count 3280 cells/mm3; differential leukocyte count was neutrophils 34, lymphocytes 66, pleural fluid triglyceride 188.0 mg%, and pleural fluid cholesterol 24.0 mg% 37]Primary treatment in the case of chylothorax should be directed toward the correction of malnutrition and compromised immunologic status which is due to repeated pleural fluid aspirations of chyle with its high levels of protein, fat, electrolytes, and lymphocytes. The defeIn our case, diagnosis of chylothorax was established on typical pleural fluid color, high pleural fluid triglyceride level, high ratio of pleural fluid to serum triglyceride, and low ratio of pleural fluid to serum cholesterol. She responded well to antituberculous treatment.Thus, we should remember that while treating any patient with chylothorax, the probable diagnosis of tuberculosis should be kept in mind.


Human tumour xenografts in immune compromised mice are widely used as cancer models because they are easy to reproduce and simple to use in a variety of pre-clinical assessments. Developments in nanomedicine have led to the use of tumour xenografts in testing nanoscale delivery devices, such as nanoparticles and polymer-drug conjugates, for targeting and efficacy via the enhanced permeability and retention (EPR) effect. For these results to be meaningful, the hyperpermeable vasculature and reduced lymphatic drainage associated with tumour pathophysiology must be replicated in the model. In pre-clinical breast cancer xenograft models, cells are commonly introduced via injection either orthotopically  or ectopically , and the organ environment experienced by the tumour cells has been shown to influence their behaviour.To evaluate xenograft models of breast cancer in the context of EPR, both orthotopic MFP and ectopic SC injections of MDA-MB-231-H2N cells were given to NOD scid gamma (NSG) mice. Animals with matched tumours in two size categories were tested by injection of a high molecular weight dextran as a model nanocarrier. Tumours were collected and sectioned to assess dextran accumulation compared to liver tissue as a positive control. To understand the cellular basis of these observations, tumour sections were also immunostained for endothelial cells, basement membranes, pericytes, and lymphatic vessels.P < 0.10). Immunostaining revealed greater vascular density and thinner basement membranes in the MFP tumour model 3 weeks after cell injection. Both the MFP and SC tumours showed evidence of insufficient lymphatic drainage, as many fluid-filled and collagen IV-lined spaces were observed, which likely contain excess interstitial fluid.SC tumours required longer development times to become size matched to MFP tumours, and also presented wide size variability and ulcerated skin lesions 6 weeks after cell injection. The 3 week MFP tumour model demonstrated greater dextran accumulation than the size matched 5 week SC tumour model (for Dextran accumulation and immunostaining results suggest that small MFP tumours best replicate the vascular permeability required to observe the EPR effect in vivo. A more predictable growth profile and the absence of ulcerated skin lesions further point to the MFP model as a strong choice for long term treatment studies that initiate after a target tumour size has been reached. Pre-clinical development of anti-cancer therapeutics relies on availability of relevant and reproducible in vivo tumour models. Human tumour xenograft models in immunodeficient mice are widely used to assess pharmacokinetics, biodistribution, and treatment efficacy because they are inexpensive and easy to replicate . HoweverProgress in nanomedicine seeks to shift distribution of therapeutic compounds to tumour tissue by targeting hyperpermeable tumour vasculature ,3. TumouSeveral pathological features of tumour vasculature lead to its utility in targeting applications. Pathological tumour vessels are dynamic, and can result both from angiogenesis and remodeling of existing vessels ,7. EndotTo validate the use of human tumour xenografts in mouse models of breast cancer to investigate tumour targeting via EPR, we studied MDA-MB-231-H2N cells transplanted in NOD scid gamma (NSG) mice and compared two common cell injection sites in the context of EPR permissive pathology. Owing to its simplicity, tumour cells are often introduced ectopically as subcutaneous (SC) injections, regardless of their native tissue type -19. CellCurrently, the benefit of using either SC or MFP in xenograft models of breast cancer in assessing targeting through the EPR effect is not well characterized. To investigate vessel permeability, orthotopic and ectopic tumour-bearing NSG mice were given intravenous injections of a fluorescently labeled high molecular weight dextran . MDA-MB-231-H2N cells and NOD scid gamma (NSG) mice were generous gifts from Dr. Robert Kerbel , which were then maintained or bred in-house. Lysine-fixable dextran-FITC (MW 2 ± 0.2 MDa) was purchased from Invitrogen . Slides and cover slips were purchased from Fisher Scientific . Primary antibodies were purchased from Abcam  for CD31 (ab28364), LYVE-1 (ab14917), collagen IV (ab19808), and αSMA (ab5694). Immunostaining reagents  were purchased from Vector Labs . Entellan hard mounting medium was purchased from EMD Millipore . All other materials were purchased from Sigma-Aldrich  and used as received unless otherwise noted.2 environment. To prepare cell suspensions for injection, adherent cells were first rinsed with phosphate buffered saline, pH 7.4 (PBS), and then incubated briefly with trypsin-ethylenediamine tetraacetic acid . Once the cells were suspended, enzymatic digestion was inhibited with FBS, and the cells were pelleted and washed 3 times in PBS before resuspension at the desired concentration. Cells were kept on ice prior to injection.MDA-MB-231-H2N cells were maintained in RPMI 1640 culture medium, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 units/mL penicillin and 50 mg/mL streptomycin under a humidified 5% COThe protocols used in these in vivo studies were approved by the University Health Network Animal Care Committee and performed in accordance with current institutional and national regulations. Animals were housed in a 12 h light and 12 h dark cycle with free access to food and water. NSG mice were bred in-house, and 7–9 week old female mice were selected for tumour xenotransplantation.6 MDA-MB-231-H2N cells suspended in 50 μL of sterile PBS. Prior to injection, mice were anaesthetized with isoflurane-oxygen. To form ectopic SC tumours, anaesthetized mice were injected with cells under the skin in the right dorsal flank. To form orthotopic mammary fat pad (MFP) tumours, the surgical area was depilated and swabbed with 70% ethanol and betadine before making an incision in the skin of the lower abdomen to the right of the midline, uncovering the mammary fat pad in the right inguinal region where cells were injected into the fat pad. The incision was then sutured closed and lactated Ringer’s solution and buprenorphine were given post-operatively for recovery and pain management. Solid tumours were allowed to form over a period of 3–5 weeks. Cohorts of tumour-bearing animals were divided into two groups to proceed onwards for testing; the first group was tested once their tumours reached an average diameter along the major axis of 7 mm as measured through the skin using calipers, and the second group tested the following week.Mice in all experimental groups were inoculated with 102 asphyxiation and tissue samples (tumour and liver) were collected by dissection; tumour samples were directly measured for diameter along both the major and minor axes (L and W) and thickness (H) using calipers  tail vein injection . After 1L × W × H), and ea2O2 in methanol for 20 min before being washed in PBS again and blocked in 1.5% normal goat serum (NGS) in PBS (see Table Three slides (six tissue sections) from each tumour were selected for each set of stains such that each slide contained sections a minimum of 300 μm away from the previous slide. Thawed slides were hydrated and washed in PBS and incubated with 0.3% HAll fluorescence images (FITC-dextran) were acquired with a fixed exposure time for each channel using an Olympus BX50 with a UPlanSApo 10×/0.40 objective, Photometrics CoolSNAP HQ2 monochrome camera, and motorized stage . Images were tiled together using Metamorph, and analyzed using ImageJ. To compensate for blood remaining in tissue after sacrifice, the blood-associated fluorescence intensity was quantified in hepatic sinusoids in liver tissue slices. Pixels matching this intensity were subtracted from the positive pixel count in the subsequent analysis.P < 0.05). Where variances were equivalent, one-way ANOVA was applied, followed by a corrected unpaired t-test; differences are denoted by square bracket symbols connecting the differing groups .Brightfield images (immunostaining) were acquired using an Aperio ScanScope XT  for whole slide scanning at 20× magnification and analyzed using ImageScope Microvessel Analysis. Statistical significance between groups was first tested with Bartlett’s test for equality of variance  and ectopically (SC) was monitored weekly through the skin in live animals using calipers. Following cell injection, MFP tumours reached a target size of 7 mm in diameter across the major axis by 3 weeks post-injection whereas SC tumours took an additional 2 weeks to reach this size. Differences in growth rate were expected, as each injection site provides a different microenvironment. Cohorts of animals were selected based on tumour size matching instead of development time because size is one of three standard measurements that determines breast cancer prognosis . After r3) has formed [P < 0.05 by Bartlett’s test of equality of variances). Unexpectedly, several tumours in the SC group at 6 weeks post-cell injection were smaller than those observed the previous week   for tranP = 0.08 by one-way ANOVA), suggesting that the orthotopic MFP environment encouraged EPR permissive vasculature and/or lymphovasculature.Based on fluorescence images of tissue sections, relatively poor dextran uptake was observed in tumour tissue compared to liver tissue across all groups. A threshold was defined to exclude background signal detected in blank tumour and liver tissue and the remaining areas, representing levels above this threshold, were quantified. Less than 1% of the positive signal area observed in the liver control was observed in tumour slices Figure . This caTo better understand the underlying vascular pathophysiology present in both tumour models, tumour slices were immunostained to provide information on the blood and lymphatic vessels present. Tissue was stained for CD31, an endothelial cell marker, to locate and characterize blood vessels. In normal blood vessels, an intact monolayer of endothelials cells is expected, whereas hyperpermeable tumour blood vessels are characterized by multiple layers of discontinuous endothelial cells that may sprout outwards or project into the vessel lumen ,13. The Separate sections were also co-stained for collagen IV to visualize the thickness of the associated basement membrane. The basement membrane forms a physical barrier that inhibits transport of high molecular weight materials across blood vessel walls ,32. In tCD31 staining also revealed differences in vascular density, with the 3 week old MFP tumours having a significantly greater vessel density than the other groups Figure . The decThe mean vascular area was also quantified, giving an indication of the size, and therefore the capacity of the blood vessels present in each tumour type. The vascular area in 3 week old MFP tumours was significantly higher than the 4 week old MFP tumours Figure , indicatAt the same time, all of the evaluated models are likely underperfused as their small size categorizes them as microvasculature . This loCD31 was also co-stained with αSMA to visualize differences in pericyte association with blood vessels. Pericytes are important blood vessel support cells that help to regulate blood flow and vessel permeability, but are often detached in tumour pathophysiology. The observed staining patterns suggest that this was the case across all tumour models Figure . PericytP < 0.05 by Bartlett’s test of equality of variances) meaning that the groups tested were not equivalent. While the mean lymphatic vessel density was highest in the 3 week old MFP tumours, the 5 week old SC tumours demonstrated the highest mean lymphatic vessel area. These factors counterbalance one another, as density and capacity each contribute to overall drainage.LYVE-1 staining was used to detect lymphatic vessels in tumour tissue. Lymphatic vessels provide a network to drain protein rich interstitial fluid back into circulation. By the nature of their function, these vessels are porous to allow macromolecules to be transported , and theThere is evidence that both the MFP and SC tumour models yielded poor lymphatic drainage compared to healthy tissue. Accumulation of interstitial fluid in cases of lymphedema has been shown to lead to the deposition of collagen . Visual Taken together, the data gathered through CD31 and collagen IV immunostaining suggest that, of the models tested, the 3 week MFP tumour best replicates the vascular permeability required to observe the EPR effect in vivo. However, the blood vessels visualized are sparse and small, contributing to low accumulation of the model nanocarrier used in this study. Both MFP and SC tumours showed evidence of excess interstitial fluid accumulation, suggesting poor lymphatic drainage in both models. While MFP tumours demonstrated greater lymphatic vessel density, SC tumours had greater lymphatic vessel size, both of which contribute to drainage, making it difficult to easily differentiate the two models in terms of drainage capacity. MFP tumours demonstrated greater utility for long-term treatment studies, as their growth is more consistent at large tumour sizes, and no skin ulcerations were observed.This study provides insight into the vascular properties of human tumour xenograft models of breast cancer in both MFP (orthotopic) and SC (ectopic) environments, two common pre-clinical models. When both animal models were challenged with a high molecular weight dextran as a model nanocarrier, there was higher accumulation in MFP tumours 3 weeks after cell injection. Further adding to the evidence that MFP tumour vasculature has greater permeability to macromolecules – a pathological feature relevant to nanocarrier accumulation via EPR – CD31 and collagen IV immunostaining revealed greater vascular density and size, as well as thinner basement membranes, in MFP tumours collected 3 weeks after cell injection. Both models demonstrated poor dextran accumulation compared to the liver as a positive control, suggesting that although several pathological features were observed, low vascular density and small blood vessel size led to relatively poor tumour perfusion. Both the MFP and SC tumour models showed evidence of poor lymphatic drainage, as several CD31 negative and collagen IV-lined fluid-filled cavities were observed. The MFP environment offered several practical benefits, including shorter development times to reach a target tumour size, more consistent growth profiles, and the absence of ulcerated skin lesions observed in SC tumour animals.α-SMA: Alpha smooth muscle actin; DAB: 3,3’-diaminobenzidine; EPR: Enhanced permeability and retention; FBS: Fetal bovine serum; LYVE-1: Lymphatic vessel endothelial hyaluronan receptor; MFP: Mammary fat pad; MMP: Matrix metalloproteinase; NGS: Normal goat serum; NSG mice: NOD scid gamma mice; PBS: Phosphate buffered saline, pH 7.4; SC: Subcutaneous.The authors declare that they have no competing interests.KSH designed the study and protocols, performed animal experiments, immunostained tissue, collected images, maintained and prepared cells for transplantation, executed the data analysis, and prepared the manuscript. PP was responsible for the breeding the mouse colony, performing cell injections, monitoring tumour growth, and assisted in designing protocols, performing the animal experiments, immunostaining tissue, and collecting images. SCO participated in designing the study and protocols, and assisted in performing SC cell injections. MSS participated in study design and was involved in writing the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/579/prepub


Tumour cells communicate with the cells of their microenvironment via a series of molecular and cellular interactions to aid their progression to a malignant state and ultimately their metastatic spread. Of the cells in the microenvironment with a key role in cancer development, tumour associated macrophages (TAMs) are among the most notable. Tumour cells release a range of chemokines, cytokines and growth factors to attract macrophages, and these in turn release numerous factors  that are implicated in invasion-promoting processes such as tumour cell growth, flicking of the angiogenic switch and immunosuppression. TAM density has been shown to correlate with poor prognosis in breast cancer, suggesting that these cells may represent a potential therapeutic target. However, there are currently no agents that specifically target TAM's available for clinical use.in vitro and in vivo models, showing that zoledronic acid can also target tumour cells to increase apoptotic cell death and decrease proliferation, migration and invasion, and that this effect is significantly enhanced in combination with chemotherapy agents. Whether macrophages in the peripheral tumour microenvironment are exposed to sufficient levels of bisphosphonate to be affected is currently unknown. Macrophages belong to the same cell lineage as osteoclasts, the major target of BPs, and are highly phagocytic cells shown to be sensitive to bisphosphonates in model studies; In vitro, zoledronic acid causes increased apoptotic cell death; in vivo the drug has been shown to inhibit the production of pro-angiogenic factor MMP-9, as well as most recent evidence showing it can trigger the reversal of the TAMs phenotype from pro-tumoral M2 to tumoricidal M1. There is thus accumulating evidence supporting the hypothesis that effects on TAMs may contribute to the anti-tumour effect of bisphosphonates. This review will focus in detail on the role of tumour associated macrophages in breast cancer progression, the actions of bisphosphonates on macrophages in vitro and in tumour models in vivo and summarise the evidence supporting the potential for the targeting of tumour macrophages with bisphosphonates.Bisphosphonates (BPs), such as zoledronic acid, are anti-resorptive agents approved for treatment of skeletal complication associated with metastatic breast cancer and prostate cancer. These agents act on osteoclasts, key cells in the bone microenvironment, to inhibit bone resorption. Over the past 30 years this has led to a great reduction in skeletal-related events (SRE's) in patients with advanced cancer and improved the morbidity associated with cancer-induced bone disease. However, there is now a growing body of evidence, both from  Breast cancer is the most commonly diagnosed cancer in the UK where women have a 1 in 8 lifetime risk of developing the disease .Cancer cells work in conjunction with cells in the surrounding microenvironment to aid numerous processes needed for tumour development. Macrophages are a major component of this microenvironment, and are of particular interest as potential therapeutic targets due to their central role in tumour progression.Macrophages are lymphocytes of the myeloid lineage, derived from CD34+ bone marrow progenitor cells  and M2 (Type II) polarised macrophages. These have different characteristics and functions within the body and immune system; shown by the varying types and amounts of cytokines they produce  are better adapted to scavenging debris, and secrete growth factors that promote angiogenesis. They show reduced immune activity such as poor antigen-presenting capabilities and suppress T cell and natural killer cell proliferation and activity. They are still highly phagocytic but mainly help repair sites of injury by engulfing cell debris, regulating tissue remodelling and repair and control normal cell turnover ,6. M2 maThe tumour stroma plays a central role in tumour progression. Via cellular and molecular interactions between the stroma and the tumour cells, the stoma is able to change with the tumour as it evolves . Cells oPrimary and secondary tumours are associated with chronic inflammation leading to recruitment of bone marrow derived cells, macrophages are a major component of this inflammatory infiltrate and thus of the tumour stroma itself ,10. Tumo2) initiates the development of TAMs, as well as initiating the development of characteristics akin to those of the M2 macrophages [Once in the tumour stroma the macrophages differentiate into tumour-associated macrophages (TAMs). It has been proposed that exposure to the tumour cells and to additional tumour-derived molecules such as IL-4, IL-10, IL-13, TGFβ-1 (transforming growth factor - β) and PGE2 , direct cell surface contact between the stromal and lymphoma cells was needed for growth regulation [in vitro when co-cultures with peritoneal macrophages [gulation . It has gulation . It woulrophages . Moreoverophages .et al described how TAMs -that secrete the majority of EGF in tumour stroma, preferentially stimulated the breast tumour cells that express EGF-receptors thereby creating a predominantly EGFR-expressing tumour, which is correlated with poor survival [A review by Leek survival . This prsurvival , supportsurvival .et al who showed reduced infiltration of macrophages into tumours in CSF-1 null mutant PyMT mice and thus, reduced invasion by tumour cells [Several reports have proposed a chemotactic and paracrine EGF/CSF-1 loop between macrophages and tumour cells, which allows them to work synergistically in an effort to co-migrate. Macrophages express CSF-1 receptors and produce EGF, whereas tumour cells express EGF receptors and produce CSF-1. EGF stimulates the migration of tumour cells and up-regulates the production of CSF-1 which subsequently promotes migration of TAMs. Thus the two cell types migrate together in a co-dependent manner. This supports findings by Lin ur cells .in vivo models of breast cancer, this migratory response is concurrent with invasion, intravasation and metastasis [Moreover, EGF and CSF-1 induce the formation of invadopodia (in metastatic mammary adenocarcinoma cells) and podosomes (in TAMs), respectively. Both are involved in extra-cellular matrix (ECM) degradation and remodelling, thus increasing invasion of both cell types -37. In iIn vitro studies have shown that co-culture of breast cancer cells and macrophages up-regulated MMP expression in macrophages in a TNF-α dependent fashion, causing enhanced invasiveness of the tumour cells [Invasion of a tumour through the basement membrane is the point at which it is classified as malignant and marks the start of the metastatic cascade. TAMs, as well as forming podosomes, are capable of secreting factors that break down areas of basement membrane, thus allowing movement of tumour cells into the surrounding tissues. These include matrix metalloproteinases in particular MMP-2, -3, -7 and -9 ,38, enzyMigration of tumour cells and TAMs occur in areas of collagen fibrillogenesis and angiogenesis, where both cell types move along collagen fibres attached to blood vessels in a lockstep fashion. Once they reach the blood vessels, macrophage aid tumour cells intravasation into the circulation. In addition, invasion occurs at sites of angiogenesis, and there is also evidence that macrophages promote collagen fibrillogenesis .In contrast to the role of macrophages in primary tumours, little is known about the specific role of TAMs in metastatic foci; the data suggests that macrophages are also involved at the other end of the metastatic cascade, aiding extravasation of tumour cells and the establishment of a proliferative niche. One study showed that when carcinoma cells were injected into the portal vein, mice with depleted peritoneal macrophages had reduced tumour foci in the lungs, indicating that the macrophages were directly involved in seeding of the tumour cells .Studies of the effects of CSF-1 on tumour progression and establishment of metastasis showed that mice deficient in macrophage CSF-1 had delayed metastatic spread of mammary tumours to the lungs. In contrast, CSF-1 deficiency did not affect development or growth of the primary tumour . Tumour 2, TGF-β1 and CSF-1, expressed by tumour cells, as well as IL-10, PGE2 and MMP-7 expressed by TAMs. IL-10, PGE2 and TGF-β have been shown to suppress the proliferation and cytotoxicity of T cells and NK cells by decreasing macrophage expression of IL-12. MMP-7 is shown to increase tumour cell resistance to chemotherapeutic agents [As discussed above M1 macrophages play important roles in both arms of the immune system including phagocytosis, antigen presentation and release of pro-inflammatory cytokines and are therefore naturally tumoricidal. In contrast, M2 macrophages show poor immune-stimulatory and antigen presenting capabilities. As would be expected, TAMs possess M2-like traits; Tumour cells and the tumour microenvironment release a variety of factors that facilitate this, including IL-4, IL-6, IL-10, PGEc agents ,43 and tc agents ,43, bothOverall the evidence suggests that the down-regulation of the normal immune-response to tumour cells by TAMs allows tumours to grow unchecked as well as aiding metastatic spread ,15.et al showed that TAM infiltration was associated with high tumour grade, tumour necrosis and large tumour size [Due to TAMs extensive involvement in tumour progression, it is no surprise that TAM infiltration of the tumour microenvironment has been correlated with decreased patient survival, especially in breast cancer. Histological analysis of 75 invasive breast cancers by Lee our size . Focal mour size . Moreoveour size .in vitro and in vivo as described in the following sections.This integral role that TAMs play in various processes aiding tumour progression and metastasis, make them an important and potential therapeutic target. One such therapy could be the commonly used anti-resorptive drugs nitrogen-containing bisphosphonates, which have been shown to affect macrophages both Bisphosphonates are stable inorganic analogues of pyrophosphonate in which the central oxygen atom has been replaced by a carbon atom (P-O-P vs P-C-P), see Figure The P-C-P structure of bisphosphonates forms the central backbone to which two side chains, R1 and R2, are covalently bonded. It is the presence or absence of nitrogen on the R2 side chain that divides bisphosphonates into their two classes; nitrogen-containing bisphosphonates (N-BPs) and non-nitrogen containing bisphosphonates (non N-BPs) -47 see .Non-nitrogen containing bisphosphonates are metabolically converted AppCl2p which inhibits the exchange between ATP and ADP, impairing mitochondrial function and thereby inducing apoptosis -50.Nitrogen containing bisphosphonates  modify protein prenylation by inhibiting farnesyldiphosphonate (FPP) synthase, a key enzyme in the mevalonate pathway, present in all eukaryotic cells -52, FiguN-BPs can further be classified into second generation N-BPs  and the more potent third generation N-BPs (zoledronic acid and risedronate). Third generation N-BPs include a heterocyclic ring at R2, i.e. a nitrogen-containing ring. Both second and third generation N-BPs are much more potent than their non-nitrogen containing predecessors, due to their increased actions on the mevalonate pathway ,47,52,53The mevalonate pathway is responsible for cholesterol synthesis and post-transitional prenylation of a number of molecules including small GTPases such as Ras (and Ras-related proteins e.g. Rap1a). By inhibiting FPP synthase, N-BPs reduce this protein prenylation, thereby inducing apoptotic cell death. They can also, indirectly, increase the synthesis of ApppI  which is converted to AMP and IPP, the excess IPP leads to apoptosis, similar to the mechanism of non N-BP's. Production of ApppI is a unique effect of N-BP's. Therefore N-BPs have two methods of inducing apoptosis; inhibition of FPP and excess production of IPP -52.Bisphosphonates are poorly absorbed in the gut due to their negative charge hindering their transport across the lipophilic cell membrane; they are therefore given mainly intravenously ,54. BPs Max) of 1-2 μM [in vitro and in vivo studies that report effects of BPs have used high concentrations and extensive incubation periods.As many studies included in this review use zoledronic acid (ZOL), it is important to note the following pharmacological information: The standard 4 mg clinical dose of ZOL administered as an infusion every 3-4 weeks in the treatment of cancer-induced bone disease has a plasma half life of 105 minutes and a peak plasma concentration  which binds to RANK receptors on pre-osteoclasts causing increased osteoclastogenesis and osteoclast activity, thus increasing bone resorption. As bone is resorbed, growth factors such as TGF-β are released which stimulate the proliferation of tumour cells, and the process continues. This process, known as the vicious cycle, is illustrated in Figure It is now well established that bisphosphonates reduce cancer-induced bone disease by interfering with this vicious cycle and osteoclast-mediated bone resorption. This in turn reduces the risk of patients developing a skeletal-related event (SRE) such as bone pain, pathological fractures and hypercalcaemia. On a molecular level, bisphosphonates reduce the release of bone derived growth factors, hence indirectly inhibiting tumour cell proliferation.in vitro but also some in vivo studies have shown N-BPs can decrease tumour cell proliferation, adhesion, migration and invasion, increase apoptosis and decrease angiogenesis. These activities not only lead to a reduction in skeletal tumour burden and hinder the progression of bone metastasis, but may also decrease tumour burden at the primary site.Evidence from primarily et al who pre-treated bovine cortical bone slices with increasing concentrations (1- 100 μM) of etidronate (ETI), clodronate (CLO), pamidronate (PAM), olpadronate, alendronate (ALN) and ibandronate (IBA) prior to seeding of MDA-MB-231 human breast cancer cells. IBA, PAM, ALN and olpadronate  all dose-dependently inhibited adhesion and spreading of breast cancer cells to bone, whereas ETI or CLO (non N-BPs) had no effect. These findings are supported studies of the effects of IBA, PAM and CLO treatment on subsequent adhesion of MDA-MB-231 and MCF-7 cells to bone, showing that the drugs reduced adhesion with the same order of potency as reported by Van der Pluijm et al [The ability of N-BPs to inhibit tumour cell adhesion in breast cancer was first reported by Van der Pluijm jm et al ,61. Bispjm et al .ZOL, CLO and IBA also showed dose-dependent inhibition of MDA-MB-231 invasion, with the order of potency of the remaining BPs was in concordance with previous results  -62.In vivo studies using murine models have reported inhibition by ZOL on invasion and migration of breast cancer cells. Female BALB/c mice injected with 4T1/luc breast cancer were treated with intravenous ZOL (5 μM/mouse). Histological examination showed a significant decrease in bone, lung and liver metastases in mice that were repeatedly treated (4 times). There was no significant increase in apoptosis of tumour cells in the lungs or the liver, indicating that ZOL had a direct effect at the primary tumour site; therefore it is possible that intensive ZOL treatment can affect migration and invasion of breast cancer cells in vivo [ in vivo .in vitro. Investigating the effects of N-BPs on viability and growth of breast cancer cell lines, Fromigue et al showed that PAM, IBA and ZOL (0.01-1000 μM) induced cell death mainly via apoptosis in MCF-7 and via necrosis in T47D cells, whereas no apoptosis was seen in MDA-MB-231 cells. PAM, IBA and ZOL also decreased cell growth in a dose- and time-dependent manner, in all cell lines (0.1-1000 μM). ZOL was found to be the fastest acting N-BP, inducing apoptosis within 2 hours (≥ 0.1 μM) in MCF-7 cells and inhibiting cell growth within 3 hours. This rapid effect may be clinically relevant as extra-skeletal exposure to BPs is very brief [et al found that ZOL and PAM reduced cell growth and viability by 50% in MDA-MB-231 and MCF-7 cells at concentrations of 15 μM and 20 μM (ZOL) and 40 μM and 30 μM (PAM). Moreover these N-BPs induced cell apoptosis in all cell lines [et al who also found that acute exposure to 100 μM of ZOL for just 2 hours caused significant induction of tumour cell apoptosis, which is again, clinically encouraging [N-BPs can also induce significant tumour cell apoptosis and inhibit cell proliferation ry brief . These fll lines . They well lines , which wouraging .et al showed that, in vitro, bisphosphonates are internalized into vesicles by fluid-phase endocytosis; once inside the cell, endosomal acidification caused the release of the bisphosphonate into the cytosol. They concluded that highly phagocytic cells such as macrophages have the ability to internalise bisphosphonates which makes them ideal targets for these drugs [in vivo, this would be dependent on the pharmacokinetic properties of the drugs, such as half-life in the circulation, as well as the location of the target cells.Macrophages and osteoclasts are highly endocytic cells that share the same lineage, so it would be logical that bisphosphonates would affect macrophages. Using macrophage like J774 cells, Thompson se drugs . Howeverin vitro, early work on mechanism of action of bisphosphonates was done on macrophage like J774 cells [Due to the difficulty of studying osteoclasts 74 cells . Bisphos74 cells ,56,68-7074 cells ,71,72. Tet al found that pamidronate, alendronate and ibandronate (100 μM/24 h) could inhibit proliferation, reduce cell viability and cause apoptotic cell death of macrophage-like J774 and RAW264 cells [in vitro.While investigating the effects of bisphosphonates on osteoclasts, Rogers 64 cells . They su64 cells ,73. ThisFurther studies explored how differences in R2 side chain of N-BPs could affect their potency, and showed that FPP synthase was inhibited by N-BPs in a dose-dependent fashion, with the following order of potency: ZOL > RIS > IBA > ALN > PAM . These fin vitro, as expected due to its superior ability to evoke ApppI production and inhibit FPP synthase.In addition to inhibiting FPP, N-BPs have the unique ability to evoke the production of ApppI, which inhibits the ability of mitochondrial ANT leading to excess IPP accumulation and consequently cell apoptosis. The order of potency of ApppI production in J774 macrophages is ZOL > RIS > IBA > ALN, whereas clodronate, a simple BP does not induce ApppI production . Taken tBisphosphonates have also been shown to affect the proliferation of macrophage precursors from bone marrow cells and bone marrow-derived macrophages (BMDMs) . BMDMs oMMP-9 is a matrix metalloproteinase produced by macrophages in response to stimulation by tumour-derived factors. It is involved in angiogenesis and tumour cell invasion, key processes in the metastatic cascade. Several studies have focussed on the effects of bisphosphonates on macrophage production of MMP-9 and how this has modified tumour progression ,71,72.et al investigated whether pamidronate or clodronate altered the regulation of MMP-9 in activated human monocyte/macrophages obtained from buffy coat cells of healthy volunteers [in vitro.Valleala lunteers . Macrophlunteers ,74. CLO in vivo. The MMP-9 - VEGF loop not only supports angiogenesis and invasion but "forces hyperactive haematopoiesis" which aids tumour progression [MMP-9 inhibition by N-BPs may also have consequences for myeloid-derived suppressor cell expansion and macrophage infiltration gression ,38. Moregression -77. In agression .in vivo, BALB-neuT mice expressing activated mouse mammary tumour virus (rat c-erbB-2/neu transgene) were treated for 5 days per week with ZOL (0.1 mg/kg) or PAM  [To investigate the effects of N-BPs on the relationship between MMP-9, TAMs and MDSCs  mg/kg,) . Treatmeet al showed that ZOL appeared to act preferentially on tumour-enhanced but not normal haematopoesis. The number of colonies in bone marrow cells in BALB/c mice treated for 16 weeks with ZOL and control mice were nearly identical. However, decrease in MMP-9 and VEGF at the tumour site did not impair angiogenesis, suggesting the presence of a bypass route e.g. up-regulation of fibroblast growth factor, although this remains to be established [Interestingly, Melani ablished . ZOL hasablished , and cerablished .Importantly, these studies provide evidence that targeting the production of MMP-9 within the tumour microenvironment is a successful alternative to trying to inhibit MMP-9 itself that has failed or resulted in toxicity ,71,72,78et al investigated the effects a single infusion of PAM could have on circulating VEGF levels over 7 days. 25 patients with advanced solid tumour and bone metastasis were infused with 90 mg of PAM and their VEGF levels were measured 1, 2 and 7 days after administration. The greatest significant decrease in VEGF levels occurred 2 days after administration and continuing up to 7 days post-administration; while these results are interesting, this study did not include a cancer-free control cohort [VEGF is a major pro-angiogenic cytokine produced by both tumour cells and TAMs and clinical studies have focussed on how N-BP's affect VEGF serum levels over time. Santini l cohort . The saml cohort . ZOL sigl cohort ,80. Simil cohort .These studies indicate that despite 4 mg of ZOL being cleared from the circulation within hours of administration, the effects on VEGF serum levels are still significant several weeks later. ZOL is slowly released from bone into the circulation over the course of around 7 days, which could account for the continued reduction of VEGF at this point. However, how ZOL is still modifying VEGF levels at day 21 and which cell types that are involved remains to be established -82. ImpoTo determine whether more frequent dosing with ZOL would cause a greater decrease in circulating VEGF, effects of repeated low-dose therapy (metronomic drug regime) on VEGF levels were investigated ,83. PatiThe reports discussed above have shown that N-BP's significantly affect three pro-angiogenic factors like MMP-9, VEGF and PDGF. However the cell type responsible is unknown, and while these factors can all be expressed by TAMs, they can also be expressed by tumour cells and other stromal cells. However, these studies important as they clearly demonstrate that treatment with ZOL alone may affect tumour growth outside the skeleton.et al investigated the cellular effects of decreased VEGF levels caused by clinically achievable doses of ZOL [in vivo following clinically relevant dosing.Using a murine model of mammary carcinoma, Coscia s of ZOL . As discs of ZOL . Histolos of ZOL ,83,84. M2MDP-LIP) [This review focuses mainly on the roles of macrophages and the effect of bisphosphonates in the tumour microenvironment in breast cancer; however effects on TAMs have also been reported in other tumours. Bisphosphonate-induced macrophage depletion has been the focus of a number of studies using animal models of metastatic lung cancer, metastatic liver cancer, melanoma and others (see Table MDP-LIP) -88.et al investigated the effects of clodrolip on macrophage infiltration in BALB/c nude mice injected with HARA-B lung cancer cell line, focusing on the effect on bone and muscles metastasis [in vitro, supporting that the effect of clodrolip on metastatic spread was due to its actions on macrophages rather than on the lung cancer cells [Hiraoka tastasis . Mice weer cells .in vitro with clodrolip, with no significant effect being observed [et al and confirms a direct relationship between depletion of TAMs and reduction in angiogenesis, tumour growth and increased survival [The effects of clodrolip combined with MCP-1 inhibition have been investigated on tumour growth and angiogenesis in a melanoma model. One day after the first clodrolip treatment (50 μl or 200 μl), athymic male NIG(S)-Nu mice were injected with human melanoma cell line IIB-MEL-J, IIB-MEL-J-MCP-1 (IIB-MEL-J cells containing a MCP-1 expression vector) or a vehicle control. Treatment continued every 5-7 days, mice were killed on day 4 or 11. Compared to control, clodrolip caused a decrease in tumour volume greater than 70% in IIB-MEL-J-MCP mice as well as increasing their survival, and this was associated with reduced tumour angiogenesis. To identify which cell types that were targeted, melanoma and endothelial cells (intrinsic for angiogenesis) were treated observed . This susurvival ,86.-1 clodrolip , clodronate dissolved in phosphate buffer (67 mM) or anti VEGF Abs 0.5 mg20 g-1 i.v. or clodrolip and anti VEGF Abs in combination. F9 mice treated with clodrolip had an 82% reduction in tumour volume, while those treated in combination had 92% reduction. Similar results were obtained in A673 mice. Immunohistochemical staining of tumours from A673 animals showed a significant reduction in blood vessel density (CD31+) , this reduction was significant up to 9 days after the end of therapy. Clodrolip-treated animals also exhibited a 93% depletion of TAMs which was not enhanced by combination treatment. As shown for other tumour cell types clodrolip had no effect on F9 or A673 in vitro, supporting that the effects on tumour volume were due to TAM depletion [TAM depletion has also been combined with an antiangiogenic therapy using VEGF neutralising antibody. Female Sv129 mice injected with F9 teratocarcinoma cells and CD-1 nude mice injected with A673 rhabdomyoscarcoma cells were treated with either 1 mg20 gepletion .The multi-targeted kinase inhibitor sorafenib has been combined with clodrolip or ZOL in models of metastatic liver cancer. The human hepatocellular cancer cell lines LM3R and SMMC7721 were orthotopically implanted into the liver of BALB/c nu/nu mice, and animals were treated with sorafenib (30 mg/kg daily), clodrolip (100 μg/kg three times per week), ZOL (100 μg/kg three times per week), or sorafenib combined with clodrolip or ZOL. Mice treated with sorafenib alone showed increased tumour macrophage infiltration and peripheral blood monocytes compared to control, however, mice receiving ZOL or clodrolip alone showed no significantly suppressed infiltration and decreased peripheral blood monocytes. Mice receiving ZOL or clodrolip alone showed no significant difference in either compared to control. Overall, combination treatment caused significantly decreased tumour growth, decreased angiogenesis, and decreased lung metastasis .The effects of ZOL differentiation of macrophages from myeloid cells to TAMs has been studies in CBA-j mice injected with AC29 mesothelioma cells . Mice rein vitro and in vivo [in vitro was determined in a number of cancer cell lines including prostate, breast, lung, pancreas and multiple myeloma. Pre-PLCaPZ NPs had highest anti-proliferative effect, and was subsequently tested in an in vivo model of prostate cancer. CD-1 nude mice were injected with human prostate cancer cells (PC-3) and divided into groups receiving either control, blank NPs, free ZOL or pre-PLCaPZ NPs (0.25 mg/ml) three times per week for three weeks. Animals treated with PLCaPZ NP showed 45% tumour weight inhibition and a tumour growth delay of 10 days, and this was significantly greater than in mice treated with free ZOL [in vivo, especially breast cancer models, would be of interest.Recent studies have aimed at optimising the delivery of ZOL using nanotechnology, in an attempt to overcome the limitations of its pharmacokinetic properties and thus extend the exposure time of extra-skeletal tumours to these agents -92. Two free ZOL . PLCaNZ free ZOL . Most infree ZOL .The combin vitro and in vivo supporting their potential anti-tumour activity, mainly from studies focussing on effects of tumour cells. However, as summarised in this review, bisphosphonates may also affect tumour growth by modifying cells of the tumour microenvironment. Bisphosphonates induce macrophage apoptosis in vitro and to inhibit the release of pro-angiogenic factors, and there is evidence showing that bisphosphonates can affect tumour macrophages in vivo by reversing their polarity to tumoricidal phenotype. Whereas data from model systems suggest that effects on macrophages in tumour microenvironment could contribute to the bisphosphonate anti-tumour effects, whether macrophages could be a target of these agents following clinical dosing remains to be determines. To firmly establish the potential for targeting of tumour macrophages with bisphosphonates requires in vivo studies using clinically relevant dosing regimens as well as analysis of TAMs in tumour material from neoadjuvant studies of patients receiving bisphosphonate treatment.Bisphosphonates are routinely used in the treatment of cancer-induced bone disease, and there is a growing body of evidence both 2: Prostaglandin E2; PyMT: Polyoma middle T oncoprotein; RANK: Receptor Activator of Nuclear Factor κ; RANKL: Receptor Activator of Nuclear Factor κ Ligand; RIS: Risedronate; SCID: Severe Combined Immunodeficiency; SRE: Skeletal Related Event; TAM: Tumour Associated Macrophages; TEM: Tumour Educated Macrophage; TGF-β: Transforming Growth Factor - Beta; TLR: Toll-like receptor; TNF-α: Tissue Necrosis Factor Alpha; VEGF: Vascular endothelial growth factor; VEGFR: Vascular endothelial growth factor receptorADP: Adenosine diphosphate; ALN: Alendronate; ANG: Angiopoietin; ApppI: New endogenous ATP analog; BP: bisphosphonate; CCL: CC chemokines; CIBD: Cancer Induced Bone Disease; CLO: Clodronate; COX2: Cyclooxygenase-2; CXCL: Chemokine Interleukin; CSF: Colony Stimulating Factor; DCIS: Ductal Carcinoma In Situ; DOX: doxorubicin; ECM: Extracellular matrix; EGF: Epidermal Growth Factor; EL: Endothelin; ETI: Etidronate; FGF2: Fibroblast Growth Factor 2; FFP: Farnesyldiphosphonate; HGF: Hepatocyte growth factor; HIF: Hypoxia-Inducible Transcription Factor; IBA: Ibandronate; IL: interleukin; IL: 1ra- IL-1 receptor antagonist; INFγ: Interferon-gamma; iNOS: Inducible NO synthase; LCIS: Lobular Carcinoma in Situ; LPS: Lipopolysaccharide; MDSC: Myeloid Derived Suppressor cell; MCP-1: Monocyte Chemotactic Protein - 1; MMPs: Matrix Metalloproteinases; NK: Natural killer cells; N-BP: Nitrogen containing bisphosphonate; NO: Nitric Oxide; PAM: Pamidronate; PDGF: Platelet-Derived Growth Factor; PGEThe authors declare they have no competing interest.TLR was the main author of the body text, carried out literature searches and designed the figures. IH conceived the idea for the manuscript, contributed to the body text and adjusted the figures. Both authors contributed equally to the work and approved the manuscript prior to submission.IH is reader in Bone Oncology at the University of Sheffield Medical School, specialising in studies of anti-cancer agents in advanced breast and prostate cancer. TLR has carried out studies of the effects of bisphosphonates on macrophages supervised by IH.