Manipulation of the tumour-associated vasculature to improve tumour therapy

Inhibition of tumour vascular growth, destruction of the tumour associated vasculature (TAV), and manipulation of the endothelial lining of the TAV provide powerful tools for anti-tumour therapy. We previously demonstrated that addition of TNF to chemotherapy improved tumour response. The major effect of TNF is an increased permeability of the tumour vascular bed resulting in augmented accumulation of co-administered drug in the tumour. As the TAV is recognised as a major candidate in tumour therapy it is becoming important to understand anti-vascular effects better. In our laboratory we examine the effect of immunotherapy on the TAV, and the effect of anti tumour-vascular therapy on tumours. This is studied in animal models, which exhibit similarities with the clinical setting, such as tumour perfusion treatment.     

Role of tumour necrosis factor in the tumour-necrotizing activity of agents with diverging toxicity

Summary We investigated the ability of various tumournecrotizing agents with diverging toxicity to induce tumour necrosis factor (TNF) and cytostatic activity inPropionibacterium-acnes-primed Swiss and tumour-bearing BALB/c mice, and the capacity of anti-TNF antibodies to inhibit induction of tumour necrosis by the agents. Lipid A and especially its combination with muramyl dipeptide induced high TNF levels in Swiss mice, as measured in the serum. Lower levels were induced by detoxified lipid A and the nontoxic dsRNA, polyadenylic polyuridylic acid, either alone or combined with muramyl dipeptide. The toxic agents also appeared the strongest inducers of mediators with cytostatic activity against cultured endothelial cells and MethA tumour cells. Anti-TNF antibodies partially reduced the cytostatic activity of the sera against MethA cells. Tumour-bearing BALB/c mice produced only low levels of TNF and cytostatic factors in response to all agents. Recombinant mouse TNF hardly reduced the DNA synthesis of MethA cells, unless normal mouse serum was added. Serum fromP.-acnes-treated Swiss mice and tumour-bearing BALB/c mice, that were inhibitory on their own, failed to potentiate the action of TNF. Serum from Swiss mice treated with toxic, but not detoxified, lipid A caused extensive tumour necrosis upon injection into MethA-bearing BALB/c mice. This activity was completely abolished by pre-incubation of the serum with anti-TNF. The tumour-necrotizing activity of the agents could be partially reduced by prior injection of these antibodies. Results show that the capacity of the agents to induce TNF and cytostatic activity is not related to their antitumour potential. Although TNF is likely to be a crucial mediator of the tumour-necrotizing action of the toxic as well as the nontoxic agents, it is probably not the sole mediator. Data also indicate that induction of tumour necrosis does not require induction of high and, thus toxic, TNF levels in the serum.     

The effect of tumour geometry on the quantification accuracy of planar 123I phantom images

IntroductionAccurate activity quantification is applied in radiation dosimetry. Planar images are important for quantification of whole-body images, enabling assessment of biodistribution from radionuclide administrations. We evaluated the effect of tumour geometry on quantification accuracy of ¹²³I planar phantom studies, including various tumour sizes, tumour-liver distances and two tumour-background ratios.Methods and materialsAn in-house manufactured abdominal phantom was equipped with a liver, different size cylindrical tumours, and a rod for tumour-liver distance variation. The geometric mean method with scatter and attenuation corrections was used for image processing. Scatter and attenuation corrections were made using the triple energy window scatter correction technique and a printed transmission sheet source, respectively. Region definitions for tumour activity distribution compensated for the partial volume effect (PVE). Activity measured in the dose calibrator served as reference for determining quantification accuracy.ResultsThe smallest tumour had the largest percentage deviation with an average activity underestimation of 34.6 ± 1.2%. Activity values for the largest tumour were overestimated by 3.1 ± 3.0%. PVE compensation improved quantification accuracy for all tumour sizes yielding accuracies of <12.4%. Scatter contribution to the tumours from the liver had minimal effect on quantification accuracy at tumour-liver distances >3 cm. With PVE compensation, increased tumour-background ratio resulted in a percentage increase of up to 26.3%.ConclusionWhen applying relevant corrections for scatter, attenuation and PVE without background activity, quantification accuracy of <13% was obtained. We demonstrated the successful implementation of a practical technique to obtain quantitative information from ¹²³I planar images.     

Clinical applications of tumour necrosis factor

Tumour necrosis factor (TNF) is a polypeptide hormone produced in vivo by activated macrophages and lymphocytes. TNF has diverse effects in vivo and has a physiological role as an immune modulator, as a mediator of the immune response, both through activation of neutrophils and eosinophils, and also affects the vascular endothelium. TNF also has antiviral activity and causes alterations in lipid metabolism. In disease states excessive production of TNF may have adverse affects. TNF has been implicated as a mediator of endotoxic shock, inflammatory joint disease, immune deficiency states, allograft rejection, and in the cachexia associated with malignant disease and some parasitic infections. When used in pharmacological doses, TNF is cytotoxic to many malignant cells in vitro and in vivo. The mechanisms underlying cytotoxicity are not fully elucidated but involve both a direct toxic effect to the cell and an indirect effect on tumour vasculature. Cytotoxicity is not universal and TNF may act as a differentiating agent or growth factor for some haematological cell types. So far the clinical application of TNF has been as a treatment for cancer in Phase I and II trials in patients with advanced disease and its efficacy here is still unproven. TNF may have potential for clinical application in combination therapy for cancer. There is experimental evidence for its interaction with other biological agents and cytotoxic drugs. The use of specific antibodies to inhibit production of TNF, or other agents to antagonise the toxic effects of TNF may have clinical relevance in counteracting septic shock and the clinical manifestations of TNF in inflammatory and neoplastic disease.     

Prompt gamma ray imaging for verification of proton boron fusion therapy: A Monte Carlo study

PurposeThe purpose of this study was to verify acquisition feasibility of a single photon emission computed tomography image using prompt gamma rays for proton boron fusion therapy (PBFT) and to confirm an enhanced therapeutic effect of PBFT by comparison with conventional proton therapy without use of boron.MethodsMonte Carlo simulation was performed to acquire reconstructed image during PBFT. We acquired percentage depth dose (PDD) of the proton beams in a water phantom, energy spectrum of the prompt gamma rays, and tomographic images, including the boron uptake region (BUR; target). The prompt gamma ray image was reconstructed using maximum likelihood expectation maximisation (MLEM) with 64 projection raw data. To verify the reconstructed image, both an image profile and contrast analysis according to the iteration number were conducted. In addition, the physical distance between two BURs in the region of interest of each BUR was measured.ResultsThe PDD of the proton beam from the water phantom including the BURs shows more efficient than that of conventional proton therapy on tumour region. A 719 keV prompt gamma ray peak was clearly observed in the prompt gamma ray energy spectrum. The prompt gamma ray image was reconstructed successfully using 64 projections. Different image profiles including two BURs were acquired from the reconstructed image according to the iteration number.ConclusionWe confirmed successful acquisition of a prompt gamma ray image during PBFT. In addition, the quantitative image analysis results showed relatively good performance for further study.     

Tumour-cell-induced production of tumour necrosis factor by monocytes of gastric cancer patients receiving BCG immunotherapy

Summary Human peripheral blood monocytes cocultured with tumour cells were used as an in vitro model of in situ interactions between tumour-infiltrating macrophages and the tumour. Tumour cells stimulated de novo expression of the human tumour necrosis factor (TNF) gene in monocytes and caused the release of TNF into the culture supernatant. A group of 14 patients with stage IVA gastric cancer receiving adjuvant chemotherapy (5-FU, Adriamycin, mitomycin C: FAM) or immunochemotherapy (BCG+FAM) was investigated for the ability of monocytes to produce TNF in vitro upon stimulation with tumour cells or purified protein derivative of tuberculin (PPD). Patients were followed at biweekly intervals, i.e. before each instillation of BCG epicutaneously over a period of 10 weeks. It was found that monocytes of some patients receiving BCG at the end of the observation period had an enhanced ability to produce TNF following stimulation with tumour cells. In contrast, such production was not substantially altered during the study period in patients on chemotherapy. PPD-induced TNF production was much weaker and was not significantly changed during this observation time. We infer that BCG immunotherapy may induce the subtle changes in some cancer patients that lead to an increased interaction between monocytes and tumour cells and result in enhanced production of cytokine(s) with antitumour properties.     

Active idiotypic vaccination in a patient with biclonal follicular lymphoma

Specific immunological responses to the idiotypic epitopes present in the surface immunoglobulin (Ig) of the clonal tumour population can be induced for active immunotherapy in patients with B-cell non-Hodgkin lymphoma (NHL). The clonality of the tumour cells should have important implications for the success of the implemented therapy. Here we report on the case of a patient enrolled in a protocol of active idiotypic immunotherapy in which previous cytofluorometric analysis showed a major IgM⁺, κp⁺ population in the tumoral cell suspensions. However, sequence analysis of both tumour sample and tumour-derived hybrids revealed the presence of two unrelated clones that used different VH and VK gene segments. It was possible to obtain hybridomas secreting these two different IgM, κp idiotypic proteins. The patient was immunised with a mixture of these two idiotypic Igs conjugated to keyhole limpet haemocyanin. Anti-idiotypic antibodies directed against both tumour-associated proteins were detected. This is the first case of anti-idiotypic therapy in a patient with a biclonal NHL. Our work calls attention to the question of clonality in the context of idiotypic vaccination in NHL patients. Received: 18 May 2000 / Accepted: 13 December 2000     

Chemotherapy of vascularised tumours: Role of vessel density and the effect of vascular “pruning”

In this work we propose to model chemotherapy taking into account the mutual interaction between tumour growth and the development of tumour vasculature. By adopting a simple model for this interaction, and assuming that the efficacy of a drug can be modulated by the vessel density, we study the constant continuous therapy, the periodic bolus-based therapy, and combined therapy in which a chemotherapic drug is associated with an anti-angiogenic agent. The model allows to represent the vessel-disrupting activity of some standard chemotherapic drugs, and shows, in the case of constant continuous drug administration, the possibility of multiple stable equilibria. The multistability suggests an explanation for some sudden losses of control observed during therapy, and for the beneficial effect of vascular “pruning” exerted by anti-angiogenic agents in combined therapy. Moreover, in case of periodic therapies in which the drug amount administered per unit time is constant (“metronomic” delivery), the model predicts a response, as a function of the bolus frequency, significantly influenced by the extent of the anti-angiogenic activity of the chemotherapic drug and by the dependence of the drug efficacy on the vessel density.     

Tumour necrosis factor and cancer

TNF is a cytokine whose diverse actions are dependent on the local microenvironment. As a member of the cytokine network, TNF plays an important role in infection and inflammation, but excessive and deregulated production can contribute to disease processes. Likewise in malignant disease, TNF may have a role in cancer therapy and contribute to host response against tumours, but it may also be involved in the progression and spread of the cancer. In experimental models, recombinant TNF can induce significant haemorrhagic necrosis, localised to the tumour vasculature and specific tumour immunity. Although the historical background and preclinical data are promising, systemic therapy with TNF in human cancer has proved highly toxic and is inactive against all tumour types so far tested. Local therapy, particularly isolated limb perfusion, has resulted in complete and long lasting tumour regressions with necrotic activity confined solely to the tumour vascular bed. However, in several animal models, TNF contributes to malignant progression and there is evidence that TNF may have autocrine or paracrine actions in human ovarian cancer.     

Unsaturated fatty acids differ between hepatic colorectal metastases and liver tissue without tumour in humans: Results from a randomised controlled trial of intravenous eicosapentaenoic and docosahexaenoic acids

IntroductionMediators derived from the n-6 polyunsaturated fatty acid (PUFA) arachidonic acid oxidation have been shown to have tumour promoting effects in experimental models, while n-3 PUFAs are thought to be protective. Here we report fatty acid concentrations in hepatic colorectal metastases compared to liver tissue without tumour in humans.MethodsTwenty patients with colorectal liver metastasis were randomized to receive a 72 h infusion of parenteral nutrition with or without n-3 PUFAs. Histological samples from liver metastases and liver tissue without tumour were obtained from 15 patients at the time of their subsequent liver resection (mean 8 days (range 4–12) post-infusion) and the fatty acid composition determined by gas chromatography.ResultsThere were no significant differences in fatty acid composition between the two intervention groups. When data from all patients were combined, liver tissue without tumour had a higher content of both n-3 and n-6 PUFAs and a lower content of oleic acid and total n-9 fatty acids compared with tumour tissue (p<0.0001, 0.0002,<0.0001 and <0.0001, respectively). The n-6/n-3 PUFA ratio was found to be higher in tumour tissue than tissue without tumour (p<0.0001).ConclusionsHepatic colorectal adenocarcinoma metastases have a higher content of n-9 fatty acids and a lower content of n-6 and n-3 PUFAs than liver tissue without tumour.     

Fluorescent Location of Human Colonic Tumour Cells by Means of an Enzyme-Inhibitor Complex

Abstract

We studied the enzymic status of the tumour cell surface protease, guanidinobenzoatase (GB) in frozen sections of a human colonic tumour grown in nude mice and also in human colons. Active enzyme was demonstrated by the binding of a synthetic fluorescent probe for the active centre of guanidinobenzoatase (GB). It was observed that tissue derived inhibitors of GB blocked the binding of this fluorescent probe and that enzyme inhibitor complex formation could be controlled by lowering the pH of the medium with lactic acid. The presence of an inhibitor of GB in the mouse tumour extract was taken advantage of by making two fluorescent derivatives of this inhibitor; both of which located GB on colonic tumour cells in frozen sections of human colon.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

The role of mechanical host-tumour interactions in the collapse of tumour blood vessels and tumour growth dynamics

A mathematical model of residual stress evolution in a growing vascular tumour is presented, in an attempt to elucidate the poorly understood phenomenon of vascular collapse. Whereas earlier studies in this area have neglected the effects of mechanical interactions between the tumour and the surrounding host tissue, the significance of these interactions for the long-term development of a tumour is now considered. The model predicts tumour stress distributions which reflect the distinctive patterns of vascular collapse reported in experimental studies. Moreover, while neglecting mechanical host/tumour interactions results in the eventual complete regression of the tumour to its avascular dormant size in the event of vascular collapse, this new model points to the possibility of oscillations in the tumour's size in the long term.     

Optimization of treatment planning for hypoxic tumours and re-modulation of radiation intensity in heavy-ion radiotherapy

AimThe purpose of this study is to optimize treatment planning in carbon ion radiotherapy, taking into account the effect of tumour hypoxia.BackgroundIn conventional hadron therapy, the goal is to create a homogenous dose in the tumour area and, thus, achieve a uniform cell survival level. Since the induction of a specific damage to cells is directly influenced by the level of hypoxia in the tissue, the varying oxygen pressure in the different regions of hypoxic tumours would disrupt the uniformity of the cell survival level.Materials and methodsUsing the Geant4 Monte Carlo Code, the physical dose profile and dose-averaged linear energy transfer were calculated in the tumour. Then, the oxygen enhancement ratio in different areas of the tumour were compared with different pressures.ResultsModulations of radiation intensities as well as energies of ion beams were calculated, both considering and disregarding the effect of hypoxia, and the required dose profiles were compared with each other. Cell survival levels were also compared between the two methods. An equation was obtained for re-modulating the beams in the presence of hypoxia, and radiation weighting factors were extracted for the beam intensities.ConclusionThe results show that taking the effect of hypoxia into account would cause the reduction of average doses delivered to the tumour tissues up to 1.54 times. In this regard, the required dose is reduced by 1.63 times in the healthy tissues before the tumour. This will result in an effective protection of healthy tissues around the tumour.     

腹部脂肪分布与2型糖尿病合并肥胖患者胰岛素抵抗和骨密度的关系研究

目的:探讨腹部脂肪分布与2型糖尿病合并肥胖患者胰岛素抵抗和骨密度的相关性。方法:选择2017年2月至2019年7月青岛大学附属医院内分泌与代谢病科收治的159例肥胖合并2型糖尿病患者(观察组)和同期100例单纯性肥胖且口服葡萄糖糖耐量试验(OGTT)正常者(对照组)。采用多层螺旋CT(MSCT)测量腹腔内脂肪面积(VFA)、腹内脂肪体积(IAFV)、全腹脂肪体积(TAV),并计算IAFV/TAV;采用全自动生化分析仪检测受试者血脂、空腹血糖(FPG)、空腹胰岛素(FINS)水平,并计算胰岛素抵抗指数(HOMA-IR);采用骨密度仪测量腰椎骨密度、股骨骨密度、髋骨骨密度。分析VFA、IAFV、TAV、IAFV/TAV与胰岛素抵抗和骨密度之间相关性。结果:观察组VFA、IAFV、IAFV/TAV、FPG、HOMA-IR、FINS、总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)高于对照组(P0.05),高密度脂蛋白胆固醇(HDL-C)、腰椎骨密度、股骨骨密度、髋骨骨密度低于对照组(P0.05),TAV与对照组比较差异无统计学意义(P0.05)。Pearson相关分析结果示VFA、IAFV、IAFV/TAV与FPG、HOMA-IR、FINS、TC、TG、LDL-C呈正相关(P0.05),与腰椎、股骨、髋骨骨密度、HDL-C呈负相关(P0.05),偏相关分析结果显示,VFA、IAFV、IAFV/TAV与HOMA-IR呈正相关(P0.05),与腰椎、股骨、髋骨骨密度呈负相关(P0.05)。结论:腹内脂肪异常堆积与2型糖尿病合并肥胖患者胰岛素抵抗和骨密度具有显著相关性,临床可通过检测腹内脂肪分布情况预估2型糖尿病合并肥胖患者发生胰岛素抵抗和骨质疏松的风险。     

Histomorphological changes in murine fibrosarcoma after hypericin-based photodynamic therapy

Histomorphological changes in murine fibrosarcoma after photodynamic therapy (PDT) based on the natural photosensitizer hypericin were evaluated. C3H/DiSn mice were inoculated with fibrosarcoma G5:1:13 cells. When the tumour reached a volume of 40-80 mm(3) the mice were intraperitoneally injected with hypericin, either in a single dose (5 mg/kg; 1 or 6 h before laser irradiation) or two fractionated doses (2.5 mg/kg; 6 and 1 h before irradiation with laser light; 532 nm, 70 mW/cm(2), 168 J/cm(2)). All groups of PDT-treated animals with single and fractionated hypericin dosing presented primary vascular reactions including vascular dilatation, congestion, thrombosis and oedema. Two hours after PDT there were necrotic changes with small, rather focal appearance. One day after therapy the necrotic areas were enhanced, often affecting a complete superficial layer of tumour tissue. Necrotic areas were accompanied with inflammation and haemorrhages.     

Development of a Novel TNFα Ligand-Receptor Binding Assay for Screening NatchemTM Libraries

Abstract

The TNFα receptor is a major therapeutic target since over production of TNFα plays a key role in the development of septic shock following bacterial infection and has been implicated in the pathogenesis of many inflammatory disorders such as rheumatoid arthritis. To screen our NATCHEM^TM Libraries for novel natural product inhibitors of this target we have developed a sensitive immobilised TNFα receptor binding assay based on a commercially available recombinant soluble TNFα receptor (p55) and [¹²⁵I]-TNFα.     

Supra-annular Valve-in-Valve implantation reduces blood stasis on the transcatheter aortic valve leaflets

Leaflet thrombosis following transcatheter aortic valve replacement (TAVR) and Valve-in-Valve (ViV) procedures has been increasingly recognized. This study aimed to investigate the effect of positioning of the transcatheter aortic valve (TAV) in ViV setting on the flow dynamics aspect of post-ViV thrombosis by quantifying the blood stasis in the intra-annular and supra-annular settings. To that end, two idealized computational models, representing ViV intra-annular and supra-annular positioning of a TAV were developed in a patient-specific geometry. Three-dimensional flow fields were then obtained via fluid-solid interaction modeling to study the difference in blood residence time (BRT) on the TAV leaflets in the two settings. At the end of diastole, a strip of high BRT ($⩾1.2s$) region was observed on the TAV leaflets in the ViV intra-annular positioning at the fixed boundary where the leaflets are attached to the frame. Such a high BRT region was absent on the TAV leaflets in the supra-annular positioning. The maximum value of BRT on the surface of non-, right, and left coronary leaflets of the TAV in the supra-annular positioning were 53%, 11%, and 27% smaller compared to the intra-annular positioning, respectively. It was concluded that the geometric confinement of TAV by the leaflets of the failed bioprosthetic valve in ViV intra-annular positioning increases the BRT on the leaflets and may act as a permissive factor in valvular thrombosis. The absence of such a geometric confinement in the ViV supra-annular positioning leads to smaller BRT and subsequently less likelihood of leaflet thrombosis.     

Deregulation of TNF expression can also cause heart valve disease

High levels of the pro-inflammatory cytokine tumour necrosis factor (TNF) have been associated with many diseases including rheumatoid arthritis (RA), ankylosing spondylitis (AS), inflammatory bowel disease (IBD) and psoriasis.Although it has been clear for twenty-five years that TNF plays a major role in RA and AS, two major questions remain unanswered: (1) What mechanism underlies the loss of control of TNF levels in patients? (2) How does TNF exert its detrimental effects? Nonetheless, biological anti-TNF drugs have become the most successful treatment of these conditions with a third of patients entering remission, and the global market for biological TNF inhibitors is now estimated at around US$35 billions. However, their use is limited by their cost, the fact that they need to be injected, non-negligible side effects and the development of resistance due to the protein (thus antigenic) nature of these TNF inhibitors. It looks inevitable that new approaches to lower the amount of TNF should be considered. To do this, a better understanding of the regulation of TNF expression is necessary.     

Quantitative autoradiography in boron neutron capture therapy considering the particle ranges in the samples

PurposeBoron neutron capture therapy is a cellular-scale particle therapy exploiting boron neutron capture reactions in boron compounds distributed in tumour cells. Its therapeutic effect depends on both the accumulation of boron in tumour cells and the neutron fluence. Autoradiography is used to visualise the micro-distribution of boron compounds.MethodsHere, we present an equation for the relationship between boron concentration and pit density on the solid-state nuclear track detector, taking into consideration the particle ranges in the samples. This equation is validated using liver-tissue sections and boron standard solutions. Moreover, we present a simple co-localisation system for pit and tissue-section images that requires no special equipment.ResultsThe equation reproduces the experimentally observed trends between boron concentration and pit density. This equation provides a theoretical explanation for the widely used calibration curve between pit density and boron concentration; it also provides a method to correct for differences of tissue-section thickness in quantitative autoradiography.ConclusionsUsing the equation together with this co-localisation system could improve micro-scale quantitative estimation in tissue sections.