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The advent of whole slide imaging has brought advanced computer-aided diagnosis via medical imaging and artificial intelligence technologies in digital pathology. The examination of tissue samples through whole slide imaging is commonly used to diagnose cancerous diseases, but the analysis of histopathology images through a decision support system is not always accurate due to variations in color caused by different scanning equipment, staining methods, and tissue reactivity. These variabilities decrease the accuracy of computer-aided diagnosis and affect the diagnosis of pathologists. In this context, an effective stain normalization method has proved as a powerful tool to standardize different color appearances and minimize color variations in histopathology images. This study reviews different stain normalization methods highlighting the main methodologies, contributions, advantages, and limitations of correlated works. The state-of-the-art methods are grouped into four distinct categories. Next, we select ten representative methods from the groups and conduct an experimental comparison to investigate the strengths and weaknesses of different methods and rank them according to selected performance accuracy measures. The quality performances of selected methods are compared in terms of quaternion structure similarity index metric, structural similarity index metric, and Pearson correlation coefficient conducting experiments on three histopathological image datasets. Our findings conclude that the structure-preserving unified transformation-based methods consistently outperform the state-of-the-art methods by improving robustness against variability and reproducibility. The comparative analysis we conducted in this paper will serve as the basis for future research, which will help to refine existing techniques and develop new approaches to address the complexities of stain normalization in complex histopathology images.

Cancer is a disease with a significant health impact: approximately 10 million cancer-related deaths occur worldwide every year. Cancer research has unraveled the diverse nature between different cancer types, patients and even within a patient or an individual tumor. Understanding the variating biology is the key in creating biomarkers to detect cancer and discover new targets for cancer drug treatment. Taking advantage of the new biomedical tools for cancer research will help to improve the efficiency and thoroughness of cancer research. Matrix metalloproteinases (MMP) are proteolytic enzymes that degrade the components of extracellular matrix, but also participate in, for example, cell signalling by processing cytokines. Many different substrates for different MMPs have been described, but we lack a complete picture of their substrate repertoires and thus their effects in healthy tissue or pathological conditions. Here, we focus on one of the MMP family members, matrix metalloproteinase 8 (MMP8), and its role in cancer, especially in oral tongue squamous cell carcinoma (OTSCC). The effect of MMP8 on cancer biology and usability depends on the cancer type, as evidenced by our systematic review. By utilizing Biobank Borealis, we showed that possible delays in sample logistics and processing lead to the degradation of blood samples and changes in MMP8 levels, highlighting the importance of well-documented sample handling. In vitro, MMP8 overexpression was shown to decrease the motility and increase cell-cell adhesion of OTSCC cells. Using terminal amine isotopic labeling of substrates (TAILS), we identified 36 novel, potential substrates of MMP8 including FXYD domain-containing ion transport regulator 5 (FXYD5). Cleavage of FXYD5, an anti-adhesive protein upregulated in many cancers, could explain the adhesion-supporting and thus motility-diminishing functions of MMP8 in OTSCC. In all, utilization of various cancer research tools enabled us to paint a fulle, Tiivistelmä Syöpä on merkittävien terveysvaikutusten sairaus: noin 10 miljoonaa syöpäkuolemaa tapahtuu vuosittain maailmanlaajuisesti. Syöpätutkimus on paljastanut eroja niin syöpätyyppien ja potilaiden välillä, kuin potilaiden ja yksittäisen kasvaimen sisällä. Muuntelevan syöpäbiologian ymmärtäminen auttaa meitä kehittämään biologisia merkkiaineita syövän tunnistamiseksi sekä löytämään uusia kohdemolekyylejä lääkehoidolle. Uusien biolääketieteellisten menetelmien hyödyntäminen parantaa syöpätutkimuksen tehokkuutta ja perusteellisuutta. Matriksin metalloproteinaasit (MMP) ovat proteolyyttisiä entsyymejä, jotka pilkkovat soluväliaineen rakenneosia, mutta ne osallistuvat myös esimerkiksi solujen väliseen kommunikaatioon sytokiinejä prosessoimalla. Eri MMP:n substraatteja on tunnistettu, mutta meiltä puuttuu hyvä kokonaiskuva monen MMP:n substraattikokonaisuuksista ja siten niiden vaikutuksista terveessä kudoksessa ja sairaustiloissa. Tässä tutkimuksessa keskitymme matriksi metalloproteinaasi 8:n (MMP8) toimintaan syövässä, erityisesti kielisyövässä. MMP8:n syöpäbiologiset vaikutukset ja käytettävyys riippuvat syöpätyypistä, kuten systemaattinen katsauksemme paljasti. Biobankki Borealista hyödyntämällä totesimme verinäytteiden mahdollisten logistiikka- ja prosessointiviiveiden vaikuttavan niiden hajoamiseen ja MMP8-tasoihin, mikä korostaa hyvin dokumentoidun näytekäsittelyn merkitystä. In vitro, MMP8:n yliekspressio vähensi kielisyöpäsolujen liikkuvuutta ja lisäsi solu-solu-adheesiota. Käyttämällä massapektometriaan pohjautuvaa TAILS-menetelmää, tunnistimme 36 uutta MMP8:n substraattia, mukaan lukien ionien siirtoa säätelevän FXYD5-proteiinin. FXYD5:n, joka yli-ilmentyy ja vähentää adheesiota useissa syövissä, pilkonta selittänee MMP8:n adheesiota lisääviä ja liikettä vähentäviä vaikutuksia kielisyövässä. Kokonaisuudessaan erilaisten syöpätutkimuksen työkalujen hyödyntäminen mahdollisti syvemmän ymmärryksen ”MMP8:n roolista syövissä”.

Submucosal invasion depth is a significant prognostic factor when assessing lymph node metastasis and cancer itself to plan proper treatment for the patient. Conventionally, oncologists measure the invasion depth by hand which is a laborious, subjective, and time-consuming process. The manual pathological examination by measuring accurate carcinoma cell invasion with considerable inter-observer and intra-observer variations is still challenging. The increasing use of medical imaging and artificial intelligence reveals a significant role in clinical medicine and pathology. In this paper, we propose an approach to study invasive behavior and measure the invasion depth of carcinoma from stained histopathology images. Specifically, our model includes adaptive stain normalization, color decomposition, and morphological reconstruction with adaptive thresholding to separate the epithelium with blue ratio image. Our method splits the image into multiple non-overlapping meaningful segments and successfully finds the homogeneous segments to measure accurate invasion depth. The invasion depths are measured from the inner epithelium edge to outermost pixels of the deepest part of particles in image. We conduct our experiments on skin melanoma tissue samples as well as on organotypic invasion model utilizing myoma tissue and oral squamous cell carcinoma. The performance is experimentally compared to three closely related reference methods and our method provides a superior result in measuring invasion depth. This computational technique will be beneficial for the segmentation of epithelium and other particles for the development of novel computer-aided diagnostic tools in biobank applications.

In the recent decade, medical image registration and fusion process has emerged as an effective application to follow up diseases and decide the necessary therapies based on the conditions of patient. For many of the considerable diagnostic analyses, it is common practice to assess two or more different histological slides or images from one tissue sample. A specific area analysis of two image modalities requires an overlay of the images to distinguish positions in the sample that are organized at a similar coordinate in both images. In particular cases, there are two common challenges in digital pathology: first, dissimilar appearances of images resulting due to staining variances and artifacts; second, large image size. In this paper, we develop algorithm to overcome the fact that scanners from different manufacturers have variations in the images. We propose whole slide image registration algorithm where adaptive smoothing is employed to smooth the stained image. A modified scale-invariant feature transform is applied to extract common information and a joint distance helps to match keypoints correctly by eliminating position transformation error. Finally, the registered image is obtained by utilizing correct correspondences and the interpolation of color intensities. We validate our proposal using different images acquired from surgical resection samples of lung cancer (adenocarcinoma). Extensive feature matching with apparently increasing correct correspondences and registration performance on several images demonstrate the superiority of our method over state-of-the-art methods. Our method potentially improves the matching accuracy that might be beneficial for computer-aided diagnosis in biobank applications.

Pro-tumorigenic activities of matrix metalloproteinase (MMP) 9 have been linked to many cancers, but recently the tumour-suppressing role of MMP9 has also been elucidated. The multifaceted evidence on this subject prompted us to examine the role of MMP9 in the behaviour of oral tongue squamous cell carcinoma (OTSCC) cells. We used gelatinase-specific inhibitor, CTT2, and short hairpin (sh) RNA gene silencing to study the effects of MMP9 on proliferation, motility and invasion of an aggressive OTSCC cell line, HSC-3. We found that the migration and invasion of HSC-3 cells were increased by CTT2 and shRNA silencing of MMP9. Proliferation, in turn, was decreased by MMP9 inhibition. Furthermore, arresten-overexpressing HSC-3 cells expressed increased levels of MMP9, but exhibited decreased motility compared with controls. Interestingly, these cells restored their migratory capabilities by CTT2 inhibition of MMP9. Hence, although higher MMP9 expression could give rise to an increased tumour growth in vivo due to increased proliferation, in some circumstances, it may participate in yet unidentified molecular mechanisms that reduce the cell movement in OTSCC.

Matrix metalloproteinases (MMPs) have traditionally been considered as tumor promoting enzymes as they degrade extracellular matrix components, thus increasing the invasion of cancer cells. It has become evident, however, that MMPs can also cleave and alter the function of various non-matrix bioactive molecules, leading to both tumor promoting and suppressive effects. We applied systematic review guidelines to study MMP8 in cancer including the use of MMP8 as a prognostic factor or as a target/anti-target in cancer treatment, and its molecular mechanisms. A total of 171 articles met the inclusion criteria. The collective evidence reveals that in breast, skin and oral tongue cancer, MMP8 inhibits cancer cell invasion and proliferation, and protects patients from metastasis via cleavage of non-structural substrates. Conversely, in liver and gastric cancers, high levels of MMP8 worsen the prognosis. Expression and genetic alterations of MMP8 can be used as a prognostic factor by examination of the tumor and serum/plasma. We conclude, that MMP8 has differing effects on cancers depending on their tissue of origin. The use of MMP8 as a prognostic factor alone, or with other factors, seems to have potential. The molecular mechanisms of MMP8 in cancer further emphasize its role as an important regulator of bioactive molecules.

The tumor microenvironment (TME) is critically important for tumor development. The microenvironment consists of fibroblasts, endothelial and immune cells as well as extracellular matrix (ECM), proteases and various other soluble factors produced by the cells. It is challenging to develop methods that appropriately mimic the human microenvironment, but this effort is essential in order to reliably elucidate the properties of potential anti-tumor drugs. The aim of this study was to create new 3D organotypic invasion models based on human tissue that would be used to study the effects of the anti-angiogenic molecules arresten and endostatin on tongue squamous carcinoma cells. The classic way to study cancer invasion has been to use a collagen invasion model that is created by mixing rat type I collagen, matrix produced by mouse EHS tumor cells and human fibroblasts. Our research group has developed a novel human myoma tissue based invasion model, which is composed of several different cell types and molecules that are normally present in the human TME. We show how this model is suitable for invasion studies, not only for oral cancer, but for other invasive cell lines as well. There are several matrix-derived fragments that have been shown to possess anti-angiogenic activity. Arresten is a 26 kDa fragment that is cleaved from type IV collagen and is known to inhibit angiogenesis, the formation of new capillaries and tumor growth in vivo. However, its effect on the tumor microenvironment in addition to endothelial cells has not been studied. We show that arresten also directly affects oral cancer cells by decreasing their migration and invasion as well as tumor size, invasion and angiogenesis in in vivo mouse xenografts. Another inhibitor of angiogenesis, endostatin, is cleaved from type XVIII collagen. It has been shown to suppress angiogenesis and tumor growth without toxicity or side effects in mouse models. Our studies show that endostatin directly affects, Tiivistelmä Syövän mikroympäristö on erittäin tärkeä syövän kehittymisen kannalta. Se koostuu fibroblasteista, endoteeli- ja immuunisoluista, soluväliaineesta, proteaaseista ja monista muista solujen tuottamista liukoisista molekyyleistä. On haastavaa kehittää uusia menetelmiä, jotka jäljittelisivät oikeaa ihmisen syövän mikroympäristöä, mutta se on välttämätöntä uusien syöpälääkkeiden tutkimiseksi. Väitöstutkimuksen tavoitteena oli kehittää kolmiulotteinen ihmisen myoomakudokseen perustuvan invaasiomalli, jonka avulla voisimme tutkia verisuonten kasvua estävien arresten ja endostatin molekyylien vaikutusta kielisyöpäsoluihin. Aiemin syövän invaasiota on tutkittu käyttämällä klassista kollageeni-invaasiomallia, joka tehdään sekoittamalla rotan tyypin I kollageeniä, hiiren sarkoomasolujen tuottamaa matriksia ja ihmisen fibroblasteja. Tutkimuksissamme kehitimme uuden invaasiomallin, joka perustuu ihmisen myoomakudokseen. Tutkimuksessa sen todettiin sisältävän monia erilaisia soluja ja molekyylejä, joita on normaalistikkin syövän mikroympäristössä. Lisäksi osoitimme, että se sopii invaasiotutkimuksiin monille syöpätyypeille. Soluvälitilamatriksista pilkotaan useita erilaisia molekyylejä joilla on osoitettu olevan angiogeneesia hillitseviä ominaisuuksia. Arresten on 26 kDa kokoinen polypeptidi, jota pilkotaan tyypin IV kollageenista. Sen tiedetään vähentävän angiogeneesia — uusien verisuonten muodostumista ja syövän kasvua in vivo. Sen vaikutuksia muihin kuin endoteelisoluihin ei ole kuitenkaan tutkittu. Tutkimuksissamme se vaikutti suoraan kielisyöpäsoluihin vähentäen niiden liikkumista ja invaasiota kolmiulotteisissa organotyyppisisssä malleissa ja hiirimallissa. Toinen tutkimamme angiogeneesin inhibiittori on endostatin, jota pilkotaan tyypin XVIII kollageenista. Sen tiedetään vähentävän angiogeneesia hiirimalleissa ilman toksisia sivuvaikutuksia. Me osoitimme tutkimuksissamme, että se vaikuttaa suoraan kielisyöpäsoluihin vähentäen niiden invaasiota ja leviämistä 3D

Cancer progression is a multistep process dependent on tumour-stroma interactions. Various cell types, such as fibroblasts, endothelial, inflammatory and stem cells, as well as extracellular matrix (ECM) proteins, such as collagens, contribute to the tumour outcome. Tumour growth and invasion is accompanied by the proteolysis of ECM components mediated by various enzymes, such as matrix metalloproteases (MMPs). Proteolytic fragments released into the circulation may reflect cancer progression. The aim of this study was to develop novel in vitro methods for investigating cell invasion and for measuring cancer-associated collagen degradation. Human carcinoma cell invasion studies in vitro are often performed in organotypic cell culture models, mainly composed of rat or mouse ECM proteins. To create a human microenvironment for invasion studies, a novel organotypic model based on human uterine myoma (benign tumour) tissue was developed. Compared to the conventional collagen-based organotypic model, in the myoma model the carcinoma cell invasion depth was about eightfold and the invasion resembled, to a greater degree, the invasion pattern of dissected tissue samples of cancer patients. In addition, the invasion was easily quantified with a novel radioimmunoassay measuring type III collagen degradation products from the organotypic culture media. As human mesenchymal stem cells (MSCs) are one of the stromal cell types that may affect tumour progression, the mechanisms of stem cell invasion were also studied. On the surface of MSCs, Toll-like receptor 9 (TLR9) functions in immune defence against microbes. The activation of TLR9 with microbial DNA-resembling molecules induced human MSC invasion into the myoma tissue in a MMP-13-mediated fashion. To analyse cancer-associated soft tissue degradation, a novel enzyme immunoassay was developed. This novel assay enabled, for the first time, the measurement of type III collagen degradation products from human serum samp, Tiivistelmä Syövän eteneminen on monivaiheinen tapahtuma, jossa syöpäsolut ovat vuorovaikutuksessa lähiympäristönsä kanssa. Ympäristön eri solutyypit, kuten kantasolut ja sidekudoksen fibroblastit sekä soluväliaineen proteiinit kuten kollageenit, vaikuttavat syöpäsolujen invaasioon eli tunkeutumiseen ympäröivään kudokseen. Syövän invaasiossa useat entsyymit, mm. matriksin metalloproteaasit (MMP:t), hajottavat soluväliainetta. Kasvaimen kehittymisen aikana verenkiertoon vapautuu soluväliaineen hajoamistuotteita, joiden määrä voi kuvastaa sairauden etenemistä. Väitöstutkimuksen tarkoituksena oli kehittää uusia menetelmiä solujen invaasion ja syöpään liittyvän kollageenin hajoamisen tutkimiseen. Ihmisen karsinoomasolujen invaasion tutkimuksessa on perinteisesti käytetty kolmiulotteisia soluviljelymalleja, jotka koostuvat pääasiassa rotan tai hiiren soluväliaineproteiineista. Työssä kehitettiin uusi viljelymalli, jossa soluja kasvatettiin ihmisen hyvälaatuisen kohtukasvainkudospalan eli myooman päällä. Perinteiseen kollageenimalliin verrattuna myoomamallissa karsinoomasolut tunkeutuivat noin kahdeksan kertaa syvemmälle, ja solujen kasvu muistutti enemmän potilaiden syöpäkudosnäytteissä havaittua kasvutapaa. Invaasion voimakkuuden määrittämiseen kehitettiin vasta-aineisiin perustuva menetelmä, jolla mitattiin soluviljelmän kasvatusliuokseen myoomakudoksesta vapautuneiden tyypin III kollageenin hajoamistuotteiden määrää. Koska kantasolujen tiedetään voivan vaikuttaa syöpäkasvaimen leviämiseen, tutkimme myös ihmisen luuytimen kantasolujen invaasiota. Kantasolujen pinnalla TLR9-reseptori osallistuu immuunipuolustukseen mikrobeja vastaan. Kun reseptoria aktivoitiin mikrobi-DNA:ta muistuttavilla molekyyleillä, kantasolut alkoivat invasoitua myoomakudokseen ja MMP-13:n aktiivisuus soluissa lisääntyi. Syöpään liittyvän pehmytkudoksen hajoamisen tutkimiseksi kehitettiin vasta-ainemenetelmä, jolla onnistuttiin ensi kertaa mittaamaan potilaiden seeruminäytteistä tyypin III kollage