Dose escalation of tolinapant (ASTX660) in combination with standard radical chemoradiotherapy in cervical cancer : a study protocol for a phase 1b TiTE-CRM clinical trial (CRAIN) in UK secondary care centres.

BACKGROUND: Cervical cancer is the fourth most common cancer in women, with an estimated 342,000 deaths worldwide in 2020. Current standard of care in the UK for locally advanced cervical cancer is concurrent chemoradiotherapy with weekly cisplatin, yet 5-year overall survival rates are only 65% with a distant relapse rate of 50%. Inhibitors of Apoptosis Proteins (IAPs) are often overexpressed in cancer cells and associated with tumour progression and resistance to treatment. Tolinapant, developed by Astex Pharmaceuticals, is an IAP antagonist with an additional mechanism of action via down-regulation of NF-kB, an important regulator in cervical cancer. Preclinical studies performed using tolinapant in combination with cisplatin and radiotherapy showed inhibition of tumour growth and enhanced survival. There is therefore a strong rationale to combine tolinapant with chemoradiotherapy (CRT). METHODS: CRAIN is a phase Ib open-label, dose escalation study to characterise the safety, tolerability and initial evidence for clinical activity of tolinapant when administered in combination with cisplatin based CRT. Up to 42 patients with newly diagnosed cervix cancer will be recruited from six UK secondary care sites. The number of participants and the duration of the trial will depend on toxicities observed and dose escalation decisions, utilising a TiTE-CRM statistical design. Treatment will constist of standard of care CRT with 45 Gy external beam radiotherapy given in 25 daily fractions over 5 weeks with weekly cisplatin 40mg/m2. This is followed by brachytherapy for which common schedules will be 28 Gy in 4 fractions high-dose-rate or 34 Gy in 2 fractions pulsed-dose-rate. Tolinapant will be administered in fixed dose capsules taken orally daily for seven consecutive days as an outpatient on alternate weeks (weeks 1, 3, 5) during chemoradiation. Dose levels for tolinapant which will be assessed are: 60 mg; 90 mg (starting level); 120 mg; 150 mg; 180 mg. Escalation will be guided by emerging safety data and decisions by the Safety Review Committee. DISCUSSION: If this trial determines a recommended phase II dose and shows tolinapant to be safe and effective in combination with CRT, it would warrant future phase trials. Ultimately, we hope to provide a synergistic treatment option for these patients to improve outcome. TRIAL REGISTRATIONS: EudraCT Number: 2021-006555-34 (issued 30th November 2021); ISRCTN18574865 (registered 30th August 2022).

Repurposing FDA approved drugs as radiosensitizers for treating hypoxic prostate cancer.

BACKGROUND: The presence of hypoxia is a poor prognostic factor in prostate cancer and the hypoxic tumor microenvironment promotes radioresistance. There is potential for drug radiotherapy combinations to improve the therapeutic ratio. We aimed to investigate whether hypoxia-associated genes could be used to identify FDA approved drugs for repurposing for the treatment of hypoxic prostate cancer. METHODS: Hypoxia associated genes were identified and used in the connectivity mapping software QUADrATIC to identify FDA approved drugs as candidates for repurposing. Drugs identified were tested in vitro in prostate cancer cell lines (DU145, PC3, LNCAP). Cytotoxicity was investigated using the sulforhodamine B assay and radiosensitization using a clonogenic assay in normoxia and hypoxia. RESULTS: Menadione and gemcitabine had similar cytotoxicity in normoxia and hypoxia in all three cell lines. In DU145 cells, the radiation sensitizer enhancement ratio (SER) of menadione was 1.02 in normoxia and 1.15 in hypoxia. The SER of gemcitabine was 1.27 in normoxia and 1.09 in hypoxia. No radiosensitization was seen in PC3 cells. CONCLUSION: Connectivity mapping can identify FDA approved drugs for potential repurposing that are linked to a radiobiologically relevant phenotype. Gemcitabine and menadione could be further investigated as potential radiosensitizers in prostate cancer.

Diffusion model comparison identifies distinct tumor sub-regions and tracks treatment response.

PURPOSE: MRI biomarkers of tumor response to treatment are typically obtained from parameters derived from a model applied to pre-treatment and post-treatment data. However, as tumors are spatially and temporally heterogeneous, different models may be necessary in different tumor regions, and model suitability may change over time. This work evaluates how the suitability of two diffusion-weighted (DW) MRI models varies spatially within tumors at the voxel level and in response to radiotherapy, potentially allowing inference of qualitatively different tumor microenvironments. METHODS: DW-MRI data were acquired in CT26 subcutaneous allografts before and after radiotherapy. Restricted and time-independent diffusion models were compared, with regions well-described by the former hypothesized to reflect cellular tissue, and those well-described by the latter expected to reflect necrosis or oedema. Technical and biological validation of the percentage of tissue described by the restricted diffusion microstructural model (termed %MM) was performed through simulations and histological comparison. RESULTS: Spatial and radiotherapy-related variation in model suitability was observed. %MM decreased from a mean of 64% at baseline to 44% 6 days post-radiotherapy in the treated group. %MM correlated negatively with the percentage of necrosis from histology, but overestimated it due to noise. Within MM regions, microstructural parameters were sensitive to radiotherapy-induced changes. CONCLUSIONS: There is spatial and radiotherapy-related variation in different models' suitability for describing diffusion in tumor tissue, suggesting the presence of different and changing tumor sub-regions. The biological and technical validation of the proposed %MM cancer imaging biomarker suggests it correlates with, but overestimates, the percentage of necrosis.

Evaluating the Efficiency of Hyaluronic Acid for Tumor Targeting via CD44.

The development of delivery systems capable of tumor targeting represents a promising strategy to overcome issues related to nonspecific effects of conventional anticancer therapies. Currently, one of the most investigated agents for cancer targeting is hyaluronic acid (HA), since its receptor, CD44, is overexpressed in many cancers. However, most of the studies on CD44/HA interaction have been so far performed in cell-free or genetically modified systems, thus leaving some uncertainty regarding which cell-related factors influence HA binding and internalization (collectively called "uptake") into CD44-expressing cells. To address this, the expression of CD44 (both standard and variants, designated CD44s and CD44v, respectively) was evaluated in human dermal fibroblasts (HDFs) and a large panel of cancer cell lines, including breast, prostate, head and neck, pancreatic, ovarian, colorectal, thyroid, and endometrial cancers. Results showed that CD44 isoform profiles and expression levels vary across the cancer cell lines and HDF and are not consistent within the cell origin. Using composite information of CD44 expression, HA binding, and internalization, we found that the expression of CD44v can negatively influence the uptake of HA, and, instead, when cells primarily expressed CD44s, a positive correlation was observed between expression and uptake. In other words, CD44shigh cells bound and internalized more HA compared to CD44slow cells. Moreover, CD44shigh HDFs were less efficient in uptaking HA compared to CD44shigh cancer cells. The experiments described here are the first step toward understanding the interplay between CD44 expression, its functionality, and the underlying mechanism(s) for HA uptake. The results show that factors other than the amount of CD44 receptor can play a role in the interaction with HA, and this represents an important advance with respect to the design of HA-based carriers and the selection of tumors to treat according to their CD44 expression profile.

Preclinical Evaluation of Ureidosulfamate Carbonic Anhydrase IX/XII Inhibitors in the Treatment of Cancers.

Carbonic anhydrases (CAs) are a family of enzymes involved in the pH regulation of metabolically active cells/tissues. Upregulation of the CAIX/XII isoforms is associated with hypoxic tumours and clinically linked with malignant progression, treatment resistance and poor prognosis. The elucidation of the crystal structure of the catalytic domains of CAIX/XII provided the basis for the generation of CAIX/XII selective inhibitors based on the sulfonamide, sulfamate and coumarins chemical structures. Ureido-substituted benzenesulfonamide CAIX/XII inhibitors have shown significant potential, with U-104 (SLC-0111) currently present in clinical Phase I/II. Ureido-substituted sulfamate CAIX/XII inhibitors have received less attention despite encouraging preclinical test results. In triple-negative breast cancer (TNBC), ureidosulfamates revealed a significant antitumour (FC9-398A) and antimetastatic potential (S4). In small cell lung cancer (SCLC), a cancer cell type very sensitive to a dysregulation in CAIX signaling, S4 treatment was particularly effective when combined with cisplatin with no evidence of acquired cisplatin-resistance. These successful anticancer strategies should provide a solid basis for future studies on ureido-substituted sulfamates.

Novel carbonic anhydrase IX-targeted therapy enhances the anti-tumour effects of cisplatin in small cell lung cancer.

Small cell lung cancer (SCLC) has an extremely poor prognosis and methods of improving chemotherapeutic intervention are much sought after. A promising approach lies in inhibiting the tumour-associated enzyme, carbonic anhydrase IX (CA IX), which supports tumour cell survival. The aim of this study was to assess the potential of CA IX inhibition using 4-(3'-(3″,5″-dimethylphenyl)ureido)phenyl sulfamate (S4), for the treatment of human SCLC alone and in combination with cisplatin chemotherapy. Treating SCLC cell lines (DMS 79 and COR-L24) with 100 µM S4 reduced viability in vitro and enhanced cell death when combined with 7 µM cisplatin, most prominently under hypoxic conditions (0.1% O2 ). When either cell line was grown as a xenograft tumour in nude mice, intraperitoneal injection of 50 mg/kg S4 alone and in combination with 3 mg/kg cisplatin led to significantly reduced tumour growth. Combination therapy was superior to single agents and response was greatly accentuated when administering repeated doses of cisplatin in DMS 79 tumours. The mechanism of therapeutic response was investigated in vitro, where S4 treatment increased apoptosis under hypoxic conditions in both DMS 79 and COR-L24 cells. DMS 79 tumours receiving S4 in vivo also displayed increased apoptosis and necrosis. Combining S4 with cisplatin reduced both the area of hypoxia and CA IX-positive cells within tumours and increased necrosis, suggesting hypoxia-specific targeting. This study presents a novel, targeted approach to improving current SCLC therapy via inhibition of CA IX, which enhances apoptosis and significantly inhibits xenograft tumour growth when administered alone and in combination with cisplatin chemotherapy.

Data-driven mapping of hypoxia-related tumor heterogeneity using DCE-MRI and OE-MRI.

PURPOSE: Previous work has shown that combining dynamic contrast-enhanced (DCE)-MRI and oxygen-enhanced (OE)-MRI binary enhancement maps can identify tumor hypoxia. The current work proposes a novel, data-driven method for mapping tissue oxygenation and perfusion heterogeneity, based on clustering DCE/OE-MRI data. METHODS: DCE-MRI and OE-MRI were performed on nine U87 (glioblastoma) and seven Calu6 (non-small cell lung cancer) murine xenograft tumors. Area under the curve and principal component analysis features were calculated and clustered separately using Gaussian mixture modelling. Evaluation metrics were calculated to determine the optimum feature set and cluster number. Outputs were quantitatively compared with a previous non data-driven approach. RESULTS: The optimum method located six robustly identifiable clusters in the data, yielding tumor region maps with spatially contiguous regions in a rim-core structure, suggesting a biological basis. Mean within-cluster enhancement curves showed physiologically distinct, intuitive kinetics of enhancement. Regions of DCE/OE-MRI enhancement mismatch were located, and voxel categorization agreed well with the previous non data-driven approach (Cohen's kappa = 0.61, proportional agreement = 0.75). CONCLUSION: The proposed method locates similar regions to the previous published method of binarization of DCE/OE-MRI enhancement, but renders a finer segmentation of intra-tumoral oxygenation and perfusion. This could aid in understanding the tumor microenvironment and its heterogeneity. Magn Reson Med 79:2236-2245, 2018. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Multi-modal imaging of long-term recovery post-stroke by positron emission tomography and matrix-assisted laser desorption/ionisation mass spectrometry.

RATIONALE: Stroke is a leading cause of disability worldwide. Understanding the recovery process post-stroke is essential; however, longer-term recovery studies are lacking. In vivo positron emission tomography (PET) can image biological recovery processes, but is limited by spatial resolution and its targeted nature. Untargeted mass spectrometry imaging offers high spatial resolution, providing an ideal ex vivo tool for brain recovery imaging. METHODS: Magnetic resonance imaging (MRI) was used to image a rat brain 48 h after ischaemic stroke to locate the infarcted regions of the brain. PET was carried out 3 months post-stroke using the tracers [18 F]DPA-714 for TSPO and [18 F]IAM6067 for sigma-1 receptors to image neuroinflammation and neurodegeneration, respectively. The rat brain was flash-frozen immediately after PET scanning, and sectioned for matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) imaging. RESULTS: Three months post-stroke, PET imaging shows minimal detection of neurodegeneration and neuroinflammation, indicating that the brain has stabilised. However, MALDI-MS images reveal distinct differences in lipid distributions (e.g. phosphatidylcholine and sphingomyelin) between the scar and the healthy brain, suggesting that recovery processes are still in play. It is currently not known if the altered lipids in the scar will change on a longer time scale, or if they are stabilised products of the brain post-stroke. CONCLUSIONS: The data demonstrates the ability to combine MALD-MS with in vivo PET to image different aspects of stroke recovery.

Tubulin-binding dibenz[c,e]oxepines: Part 2. Structural variation and biological evaluation as tumour vasculature disrupting agents.

5,7-Dihydro-3,9,10,11-tetramethoxybenz[c,e]oxepin-4-ol 1, prepared from a dibenzyl ether precursor via Pd-catalysed intramolecular direct arylation, possesses broad-spectrum in vitro cytotoxicity towards various tumour cell lines, and induces vascular shutdown, necrosis and growth delay in tumour xenografts in mice at sub-toxic doses. The biological properties of 1 and related compounds can be attributed to their ability to inhibit microtubule assembly at the micromolar level, by binding reversibly to the same site of the tubulin αß-heterodimer as colchicine 2 and the allocolchinol, N-acetylcolchinol 4.

Monocarboxylate Transporter 1 (MCT1) is an independent prognostic biomarker in endometrial cancer.

BACKGROUND: Endometrial cancer (EC) is a major health concern due to its rising incidence. Whilst early stage disease is generally cured by surgery, advanced EC has a poor prognosis with limited treatment options. Altered energy metabolism is a hallmark of malignancy. Cancer cells drive tumour growth through aerobic glycolysis and must export lactate to maintain intracellular pH. The aim of this study was to evaluate the expression of the lactate/proton monocarboxylate transporters MCT1 and MCT4 and their chaperone CD147 in EC, with the ultimate aim of directing future drug development. METHODS: MCT1, MCT4 and CD147 expression was examined using immunohistochemical analysis in 90 endometrial tumours and correlated with clinico-pathological characteristics and survival outcomes. RESULTS: MCT1 and MCT4 expression was observed in the cytoplasm, the plasma membrane or both locations. CD147 was detected in the plasma membrane and associated with MCT1 (p = 0.003) but not with MCT4 (p = 0.207) expression. High MCT1 expression was associated with reduced overall survival (p = 0.029) and remained statistically significant after adjustment for survival covariates (p = 0.017). CONCLUSION: Our data suggest that MCT1 expression is an important marker of poor prognosis in EC. MCT1 inhibition may have potential as a treatment for advanced or recurrent EC.

Targeting tumour hypoxia to prevent cancer metastasis. From biology, biosensing and technology to drug development: the METOXIA consortium.

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O(2)), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.

Transmission FT-IR chemical imaging on glass substrates: applications in infrared spectral histopathology.

Fourier transform-infrared (FT-IR) chemical imaging in transmission mode has traditionally been performed on expensive mid-IR transparent windows such as barium/calcium fluoride, which are more fragile than glass, making preparation in the histopathology laboratories more cumbersome. A solution is presented here by using cheap glass substrates for the FT-IR chemical imaging, which has a high-wavenumber transmission window allowing measurement of the C-H, N-H, and O-H stretches occurring at ca. 2500-3800 cm(-1). The "fingerprint" region of the IR spectrum occurring below 1800 cm(-1) is not obtainable; however, we demonstrate that a wealth of information is contained in the high wavenumber range using 71 patients on a breast tissue microarray (TMA) as a model for investigation. Importantly, we demonstrate that the tissue can be classified into four basic tissue cell types and that using just the epithelial cells, reasonable discrimination of normal and malignant tissue can be found.

The radiosensitising effect of gemcitabine and its main metabolite dFdU under low oxygen conditions is in vitro not dependent on functional HIF-1 protein.

BACKGROUND: Regions within solid tumours often experience oxygen deprivation, which is associated with resistance to chemotherapy and irradiation. The aim of this study was to evaluate the radiosensitising effect of gemcitabine and its main metabolite dFdU under normoxia versus hypoxia and to determine whether hypoxia-inducible factor 1 (HIF-1) is involved in the radiosensitising mechanism. METHODS: Stable expression of dominant negative HIF-1α (dnHIF) in MDA-MB-231 breast cancer cells, that ablated endogenous HIF-1 transcriptional activity, was validated by western blot and functionality was assessed by HIF-1α activity assay. Cells were exposed to varying oxygen environments and treated with gemcitabine or dFdU for 24 h, followed by irradiation. Clonogenicity was then assessed. Using radiosensitising conditions, cells were collected for cell cycle analysis. RESULTS: HIF-1 activity was significantly inhibited in cells stably expressing dnHIF. A clear radiosensitising effect under normoxia and hypoxia was observed for both gemcitabine and dFdU. No significant difference in radiobiological parameters between HIF-1 proficient and HIF-1 deficient MDA-MB-231 cells was demonstrated. CONCLUSIONS: For the first time, radiosensitisation by dFdU, the main metabolite of gemcitabine, was demonstrated under low oxygen conditions. No major role for functional HIF-1 protein in radiosensitisation by gemcitabine or dFdU could be shown.

Detection of apoptosis by PET/CT with the diethyl ester of [¹8F]ML-10 and fluorescence imaging with a dansyl analogue.

The diethyl ester of [(18)F]ML-10 is a small molecule apoptotic PET probe for cancer studies. Here we report a novel multi-step synthesis of the diethyl ester of ML-10 in excellent yields via fluorination using Xtal-Fluor-E. In addition, a one-pot radiosynthesis of the diethyl ester of [(18)F]ML-10 from nucleophilic [(18)F]fluoride was completed in 23% radiochemical yield (decay corrected). The radiochemical purity of the product was ≥99%. The diethyl ester of [(18)F]ML-10 was used in vivo to detect apoptosis in the testes of mice. In parallel studies, the dansyl-ML-10 diethyl ester was prepared and used to detect apoptotic cells in an in vitro cell based assay.

Dose-dependent changes in cardiac function, strain and remodelling in a preclinical model of heart base irradiation.

BACKGROUND AND PURPOSE: Radiation induced cardiotoxicity (RICT) is as an important sequela of radiotherapy to the thorax for patients. In this study, we aim to investigate the dose and fractionation response of RICT. We propose global longitudinal strain (GLS) as an early indicator of RICT and investigate myocardial deformation following irradiation. METHODS: RICT was investigated in female C57BL/6J mice in which the base of the heart was irradiated under image-guidance using a small animal radiation research platform (SARRP). Mice were randomly assigned to a treatment group: single-fraction dose of 16 Gy or 20 Gy, 3 consecutive fractions of 8.66 Gy, or sham irradiation; biological effective doses (BED) used were 101.3 Gy, 153.3 Gy and 101.3 Gy respectively. Longitudinal transthoracic echocardiography (TTE) was performed from baseline up to 50 weeks post-irradiation to detect structural and functional effects. RESULTS: Irradiation of the heart base leads to BED-dependent changes in systolic and diastolic function 50 weeks post-irradiation. GLS showed significant decreases in a BED-dependent manner for all irradiated animals, as early as 10 weeks after irradiation. Early changes in GLS indicate late changes in cardiac function. BED-independent increases were observed in the left ventricle (LV) mass and volume and myocardial fibrosis. CONCLUSIONS: Functional features of RICT displayed a BED dependence in this study. GLS showed an early change at 10 weeks post-irradiation. Cardiac remodelling was observed as increases in mass and volume of the LV, further supporting our hypothesis that dose to the base of the heart drives the global heart toxicity.

Endothelial heparan sulfate 6-O-sulfation levels regulate angiogenic responses of endothelial cells to fibroblast growth factor 2 and vascular endothelial growth factor.

Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor 165 (VEGF(165)) are potent pro-angiogenic growth factors that play a pivotal role in tumor angiogenesis. The activity of these growth factors is regulated by heparan sulfate (HS), which is essential for the formation of FGF2/FGF receptor (FGFR) and VEGF(165)/VEGF receptor signaling complexes. However, the structural characteristics of HS that determine activation or inhibition of such complexes are only partially defined. Here we show that ovarian tumor endothelium displays high levels of HS sequences that harbor glucosamine 6-O-sulfates when compared with normal ovarian vasculature where these sequences are also detected in perivascular area. Reduced HS 6-O-sulfotransferase 1 (HS6ST-1) or 6-O-sulfotransferase 2 (HS6ST-2) expression in endothelial cells impacts upon the prevalence of HS 6-O-sulfate moieties in HS sequences, which consist of repeating short, highly sulfated S domains interspersed by transitional N-acetylated/N-sulfated domains. 1-40% reduction in 6-O-sulfates significantly compromises FGF2- and VEGF(165)-induced endothelial cell sprouting and tube formation in vitro and FGF2-dependent angiogenesis in vivo. Moreover, HS on wild-type neighboring endothelial or smooth muscle cells fails to restore endothelial cell sprouting and tube formation. The affinity of FGF2 for HS with reduced 6-O-sulfation is preserved, although FGFR1 activation is inhibited correlating with reduced receptor internalization. These data show that 6-O-sulfate moieties in endothelial HS are of major importance in regulating FGF2- and VEGF(165)-dependent endothelial cell functions in vitro and in vivo and highlight HS6ST-1 and HS6ST-2 as potential targets of novel antiangiogenic agents.

Carbonic anhydrase IX as a target for metastatic disease.

Metastatic disease is responsible for the majority of cancer related deaths. Tumour-associated carbonic anhydrase IX (CA IX) is a powerful marker to diagnose various types of metastatic cancers including those of cervical, renal, breast and head & neck origin. The precise prognostic role of CA IX in determining local control versus overall survival is complex, although the majority of reports favour CA IX as a marker for poor prognosis in patients with metastatic cancer. Preclinical studies in cell cultures clearly demonstrate that CA IX stimulates features that enhance metastatic properties of cancer cells for example, reducing cell adhesion, increasing motility and migration, inducing vascularisation and activating proteases, in which CA IX-induced acidification of the microenvironment of the tumour is essential. As most findings are consistent with the idea that CA IX is important in metastatic dissemination, small molecular CA IX inhibitors (including fluorescent-tagged or radiolabelled) and monoclonal antibodies targeting the CA IX isoform have been developed. Studies in tumour xenograft models showed that these CA IX-specific inhibitors and antibodies can be very effective in therapy and imaging of a variety of different metastatic cancers.

Inhibition of carbonic anhydrase activity modifies the toxicity of doxorubicin and melphalan in tumour cells in vitro.

Carbonic anhydrase IX (CA IX) is a hypoxia-regulated enzyme, overexpressed in many types of human cancer. CA IX is involved in pH homeostasis, contributing to extracellular acidification and tumourigenesis. Acidification of the extracellular milieu can impact upon cellular uptake of chemotherapeutic drugs by favouring weak acids (e.g. melphalan), but limiting access of weak bases (e.g. doxorubicin). We investigated whether alterations of CA IX activity affected anti-cancer drug uptake and toxicity. CA inhibitor acetazolamide (AZM) enhanced doxorubicin toxicity but reduced melphalan toxicity in cell lines that highly expressed CA IX under anoxic conditions (HT29 and MDA435 CA9/18). The toxicity changes reflected modification of passive drug uptake. AZM did not alter toxicity or uptake in cells with low CA IX activity (HCT116 and MDA435 EV1). AZM lowered intracellular pH in HT29 and MDA435 CA9/18 cells under anoxic conditions. CA IX activity has chemomodulatory properties and is an attractive target for anti-cancer therapy.

Invasion and Secondary Site Colonization as a Function of In Vitro Primary Tumor Matrix Stiffness: Breast to Bone Metastasis.

Increased breast tissue stiffness is correlated with breast cancer risk and invasive cancer progression. However, its role in promoting bone metastasis, a major cause of mortality, is not yet understood. It is previously identified that the composition and stiffness of alginate-based hydrogels mimicking normal (1-2 kPa) and cancerous (6-10 kPa) breast tissue govern phenotype of breast cancer cells (including MDA-MB-231) in vitro. Here, to understand the causal effect of primary tumor stiffness on metastatic potential, a new breast-to-bone in vitro model is described. Together with alginate-gelatin hydrogels to mimic breast tissue, 3D printed biohybrid poly-caprolactone (PCL)-composite scaffolds, decellularized following bone-ECM deposition through Saos-2 engraftment, are used to mimic the bone tissue. It is reported that higher hydrogel stiffness results in the increased migration and invasion capacity of MDA-MB 231 cells. Interestingly, increased expression of osteolytic factors PTHrP and IL-6 is observed when MDA-MB-231 cells pre-conditioned in stiffer hydrogels (10 kPa, 3% w/v gelatin) colonize the bone/PCL scaffolds. The new breast-to-bone in vitro models herein described are designed with relevant tissue microenvironmental factors and could emerge as future non-animal technological platforms for monitoring metastatic processes and therapeutic efficacy.

Spatial Gene Expression Changes in the Mouse Heart After Base-Targeted Irradiation.

PURPOSE: Radiation cardiotoxicity (RC) is a clinically significant adverse effect of treatment for patients with thoracic malignancies. Clinical studies in lung cancer have indicated that heart substructures are not uniformly radiosensitive, and that dose to the heart base drives RC. In this study, we aimed to characterize late changes in gene expression using spatial transcriptomics in a mouse model of base regional radiosensitivity. METHODS AND MATERIALS: An aged female C57BL/6 mouse was irradiated with 16 Gy delivered to the cranial third of the heart using a 6 × 9 mm parallel opposed beam geometry on a small animal radiation research platform, and a second mouse was sham-irradiated. After echocardiography, whole hearts were collected at 30 weeks for spatial transcriptomic analysis to map gene expression changes occurring in different regions of the partially irradiated heart. Cardiac regions were manually annotated on the capture slides and the gene expression profiles compared across different regions. RESULTS: Ejection fraction was reduced at 30 weeks after a 16 Gy irradiation to the heart base, compared with the sham-irradiated controls. There were markedly more significant gene expression changes within the irradiated regions compared with nonirradiated regions. Variation was observed in the transcriptomic effects of radiation on different cardiac base structures (eg, between the right atrium [n = 86 dysregulated genes], left atrium [n = 96 dysregulated genes], and the vasculature [n = 129 dysregulated genes]). Disrupted biological processes spanned extracellular matrix as well as circulatory, neuronal, and contractility activities. CONCLUSIONS: This is the first study to report spatially resolved gene expression changes in irradiated tissues. Examination of the regional radiation response in the heart can help to further our understanding of the cardiac base's radiosensitivity and support the development of actionable targets for pharmacologic intervention and biologically relevant dose constraints.